Electrospray catheter

ABSTRACT

An apparatus includes a catheter, and an electrode and methods of delivering molecules to eukaryotic cells using such an apparatus. The catheter defines a fluidic channel and has a distal opening. The electrode is within the fluidic channel and is spaced a distance from the distal opening of the catheter. The catheter is arranged to prevent direct contact between any electrode of the apparatus and tissue. Related apparatus, systems, techniques and articles are also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a national stage application filed under 35 U.S.C. §371, of International Patent Application No. PCT/IB2018/000831 filedJun. 29, 2018 which claims the benefit of and priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62/527,989 filed on Jun. 30,2017, the entire contents of each of which are hereby expresslyincorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter described herein relates to an electrospray catheterand targeted delivery of fluids using the electrospray catheter.

BACKGROUND

Lung cancer is accountable for the highest number of cancer deathsworldwide, with poor survival rates despite advances in chemotherapyover recent years. The most effective method to substantially improvesurvival figures would be diagnosing lung cancer at an earlier stage,when therapy may be more effective.

Lung cancer has still a very poor prognosis compared to other types ofcancer. Three studies have been reported to date with mixed findingsregarding the efficacy of methylene blue staining for identification ofmalignant and premalignant lesions in a prospective manner. In thesestudies, relatively large volumes of methylene blue were delivered intothe airways using conventional spray catheters. The first study byOvchinnikov (Ovvhinnikov A A, Dianov V V, Lukomosky G I.Chromobronchoscopy in the Diagnosis of Bronchial Tumours. Endoscopy.1980; 12:147-150) delivered 2 ml of 0.2-0.4% methylene blue using abronchoscopic atomizer and reported successful differential staining ofmetaplasia/tumors versus normal tissue. The second study by Varoli(Varoli F, Mariani C, Fasceanella A, Cosentino F, Vital Staining inFibreoptic Bronchoscopy. Endoscopy. 1986; 18:142-143) delivered 0.7%methylene blue (the volume was not reported) using a spray catheter andreported that normal bronchial mucosa did not stain while malignantneoplastic tissue stained darkly and areas of squamous metaplasiastained lightly. The third more recent study by Zirlik (Zirlik S,Hildner K M, Neurath M F, Fuchs F S. Methylene blue-aided in vivostaining of central airways during flexible bronchoscopy. ScientificWorld Journal. 2012; 2012:625867. doi:10.1100/2012/625867) delivered 10ml of 0.1% methylene blue using a spray catheter. They reported thatwhile methylene blue was an appropriate dye for chromobronchoscopy andthat there were sufficient experiences on topical pulmonary application,the technique they used was not helpful for early detection of malignantor premalignant lesions of the bronchial system. It is hypothesized thatthe large volumes of methylene blue used in these studies, along withthe use of spray catheters, which do not allow controlled, targeteddelivery, contributed significantly to irreproducibility.

SUMMARY

In an aspect, an apparatus includes a catheter, and an electrode. Thecatheter defines a fluidic channel and has a distal opening. Theelectrode is within the fluidic channel and is spaced a distance fromthe distal opening of the catheter. The catheter is arranged to preventdirect contact between any electrode of the apparatus and tissue.

One or more of the following features can be included in any feasiblecombination. For example, a conductive sheath can be included on anexterior of the catheter and can be configured to couple to a ground.The catheter can separate the fluidic channel and the conductive sheath.A biocompatible cover enclosing the conductive sheath between thebiocompatible cover and the catheter can be included. The biocompatiblecover can extend less than an entire length of the catheter, and adistal end of the catheter can be exposed. The catheter can have aninner diameter of about 0.5 mm and an outer diameter of about 1.4 mm.The distance between the electrode and the distal opening can be about100 mm. The biocompatible cover can have an exterior diameter of about2.05 mm. The catheter can extend about 50 mm beyond the biocompatiblecover at the distal end.

A wire extending through a portion of a length of the catheter andwithin the fluidic channel can be included. The wire can include aninsulation layer and an exposed distal portion. The exposed distalportion can form the electrode. The electrode, when carrying a charge,can impart the charge to fluid traveling through the fluidic channel.The charge can be imparted within the fluidic channel so that, whencharged fluid reaches a fluid/air interface near the distal opening ofthe catheter, the charged fluid forms charged droplets that disperse.

The catheter can include a porous tip disposed within the fluidicchannel. The porous tip can include a felt material and/or a fibrousmaterial. The porous tip can include a cone shape geometry. The cathetercan include a protrusion disposed near the distal opening of thecatheter and surrounding an outer surface of the catheter; and athermoplastic wrap that encloses the protrusion to provide sealing andelectrical isolation.

In another aspect, a system can include a pump, a power supply, acatheter, and an electrode. The catheter is coupled to the pump, definesa fluidic channel, and has a distal opening. The electrode iselectrically coupled to the power supply, within the fluidic channel,and spaced a distance from the distal opening of the catheter. Thecatheter prevents direct contact between any electrode of the system andtissue.

One or more of the following features can be included in any feasiblecombination. For example, the pump can meter between 1 and 10microliters of fluid to the fluidic channel per actuation of the pump.The pump can meter between 5 and 500 microliters of fluid to the fluidicchannel per actuation of the pump. The power supply charges theelectrode to between 3 kilovolts and 10 kilovolts. The power supply cansupply charge to the electrode at greater than 160 nanoamps and lessthan 25 microamps.

A conductive sheath can be included on an exterior of the catheter andcan be configured to couple to a ground. The catheter can separate thefluidic channel and the conductive sheath. A biocompatible coverenclosing the conductive sheath between the biocompatible cover and thecatheter can be included. The biocompatible cover can extend less thanan entire length of the catheter, and a distal end of the catheter canbe exposed. The catheter can have an inner diameter of about 0.5 mm andan outer diameter of about 1.4 mm. The distance between the electrodeand the distal opening can be about 100 mm. The biocompatible cover canhave an exterior diameter of about 2.05 mm. The catheter can extendabout 50 mm beyond the biocompatible cover at the distal end.

A wire extending through a portion of a length of the catheter andwithin the fluidic channel can be included. The wire can include aninsulation layer and an exposed distal portion. The exposed distalportion can form the electrode. The electrode, when carrying a charge,can impart the charge to fluid traveling through the fluidic channel.The charge can be imparted within the fluidic channel so that, whencharged fluid reaches a fluid/air interface near the distal opening ofthe catheter, the charged fluid forms charged droplets that disperse.

In yet another aspect, an electrode within a fluidic channel defined bya catheter having a distal opening is charged. The electrode is spacedfrom the distal opening by a distance. The catheter is arranged toprevent direct contact between any electrode and tissue. A meteredvolume of fluid is supplied to the fluidic channel to deliver fluid to atarget.

One or more of the following features can be included in any feasiblecombination. The electrode, when carrying a charge, can impart thecharge to fluid traveling through the fluidic channel, the chargeimparted within the fluidic channel so that, when charged fluid reachesa fluid/air interface near the distal opening of the catheter, thecharged fluid forms charged droplets that disperse. The fluid caninclude Methylene Blue. The catheter can be inserted into an airway of apatient for delivering the fluid to target lung tissue. The catheter canbe inserted into one or more of: gastrointestinal tract, oral cavity,central airway, proximal airway, small intestine, and colon. The fluidcan be delivered to the target, the target including a portion of one ormore of: bowel tissue, parathyroid gland, sentinel node, melanoma, oraltissue, and small intestine tissue. The catheter can be inserted into abronchealscope.

The fluid can contain Deoxyribonucleic acid (DNA), messenger Ribonucleicacid (mRNA), small interfering Ribonucleic acid (siRNA), and/or protein,which are delivered to the target. The fluid can contain messengerRibonucleic acid (mRNA).

A conductive sheath can be included on an exterior of the catheter andcan be configured to couple to a ground. The catheter can separate thefluidic channel and the conductive sheath. A biocompatible coverenclosing the conductive sheath between the biocompatible cover and thecatheter can be included. The biocompatible cover can extend less thanan entire length of the catheter, and a distal end of the catheter canbe exposed. The catheter can have an inner diameter of about 0.5 mm andan outer diameter of about 1.4 mm. The distance between the electrodeand the distal opening can be about 100 mm. The biocompatible cover canhave an exterior diameter of about 2.05 mm. The catheter can extendabout 50 mm beyond the biocompatible cover at the distal end.

A wire extending through a portion of a length of the catheter andwithin the fluidic channel can be included. The wire can include aninsulation layer and an exposed distal portion. The exposed distalportion can form the electrode. The electrode, when carrying a charge,can impart the charge to fluid traveling through the fluidic channel.The charge can be imparted within the fluidic channel so that, whencharged fluid reaches a fluid/air interface near the distal opening ofthe catheter, the charged fluid forms charged droplets that disperse.

The fluid can contain ethanol at greater than 5% concentration. Thefluid can contain ethanol at a concentration between 5 and 75%. Thefluid can contain ethanol at about 5, 10, 15, 20, 25, 30, 25, 40, 45,50, 55, 60, 65, 70, or 75 percent. About can be within 10%, 5%, 4% 3%,2%, or 1%. For example, the ethanol concentration in the delivery fluidis greater than 5% and less than 80, 75, 70, 65, 60, 55, 50, 45, 40, 35,30, or 25% ethanol. An exemplary delivery fluid contains ethanol, aphysiologically-acceptable buffer solution, and a molecule of interestto be delivered into a viable mammalian cell, e.g., a human cell. Themolecule to be delivered comprises a nucleic acid such as a messengerRNA or Small (or short) interfering RNA (siRNA) or a protein.

A distal end of the catheter can be positioned about 15 mm from asurface of tissue.

Delivery of fluid to the target can be performed for between 0.1 secondand 10 minutes. The delivery of fluid to the target can be performed forabout 0.1 seconds, 0.2 seconds, 1 second, 10 seconds, 1 minute, 2minutes, 3 minutes, 4 minutes, or 10 minutes. Delivery of fluid to thetarget can be performed by a single spray. Delivery of fluid to thetarget can be performed with a flow rate of about 0.1 μl per minute, 3μl per minute, 4.8 μl per minute 10 μl per minute, 100 μl per minute, or1 ml per minute.

The charging can be to a voltage between 3 kV and 5 kV. The charging canbe to a voltage of about 3 kV, wherein about is within 10%. The distalopening can be 22, 23, 25, 27, 30, or 32 gauge. The distal opening canbe 32 gauge. Positive mode electrospray can be utilized.

The current subject matter includes a platform for targeted delivery ofdiagnostic and therapeutic agents to anatomical locations or organscomprising a lumen, e.g., a lumen through which a catheter is passed.Examples include the pulmonary tissues (including the lung, bronchi andbronchioles), esophagus, urinary bladder (including urethra), stomach,intestines, cardiovascular tissues as well other tissues. In someaspects, the current subject matter includes spray delivery platformtechnology enabling delivery of diagnostic and therapeutic moleculesdirectly to sites in the body in a targeted and controllable manner. Thevolume of fluid delivered can be small as compared to some exitingcatheters and the fluid can be delivered with a diverse spray.

By way of illustration, in vivo experiments in pigs were performed andare described herein, which supported the efficacy and safety of thecurrent subject matter. Furthermore, experiments using ex vivo lungcancer tissue to show a differential uptake of methylene blue dye incancer compared to healthy tissue were also performed and are describedherein. The ex vivo models described herein represent art-recognizedmodels for in vivo applications and therapy.

In an example implementation, the platform includes an ‘electrospray’catheter device designed for use in the lungs in vivo, enabling deliveryof diagnostic and therapeutic molecules directly to tissues in vivo. Thecatheter can be deployed using a conventional flexible endoscope.Physicians working in cancer, targeted drug delivery, gene therapy, anddiagnostics can use the catheter. The rationale underlying this spraycatheter approach is that a targeted, physical mode of delivery canempower clinicians to enhance patient care by (A) enabling noveldiagnostic approaches, (B) delivering agents that cannot easily bedelivered by existing delivery modes and (C) circumventing problems andlimitations associated with aerosol mediated, viral and liposome vectordelivery.

The subject matter described herein provides many technical advantages.For example, the current subject matter can deliver small amount offluid (e.g., between 1 and 10 micro liters), which can cover amicroscopically visible area in vivo. Controlled delivery allows forbetter targeting of tissue for delivery, reduced trauma to tissue,little or no localized pressure on tissue or enclosed area (e.g., vesselor lumen). Improved localized delivery allows for targeting specifictissue such as lesion, with minimal collateral damage while deliveringmore payload to the target. The current subject matter limits exposureof the patient and target tissue to electrical charge so that only theonly charge exposed to the patient and/or tissue is through the fluid,which causes the fluid to disperse when it exits the catheter distalend.

In some implementations, vector-free delivery of molecules can beachieved. The molecules can be delivered not only onto, but intotissues.

The current subject matter can differentially stain lung cancer comparedto normal “healthy” tissue in humans. This allows for improved targetingof lung biopsies during bronchoscopy. This improves the sensitivity ofbiopsies in providing an earlier lung cancer diagnosis and avoiding theneed for the patient to progress to further, more invasive, proceduresto confirm a diagnosis.

The platform can enhance patient care, offering clinicians an additionalstrategy for the diagnosis and treatment of lung disease complimentingexisting intravenous and nebulized therapy. In particular, the currentsubject matter can enable clinicians to deliver molecules, such as DNAand siRNA, in the setting of lung disease and to address a broad rangeof other disease processes outside of the lung.

Molecules to be delivered to eukaryotic cells, e.g., mammalian cellssuch as human cells are purified. As used herein, an “isolated” or“purified” nucleotide or polypeptide (e.g., a nucleotide or polypeptide)is substantially free of other nucleotides and polypeptides with whichthe cargo molecule exists in nature. Purified nucleotides andpolypeptides are also free of cellular material or other chemicals whenchemically synthesized. Purified compounds are at least 60% by weight(dry weight) the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. For example, a purifiednucleotides and polypeptides, e.g., a chemoattractant, cytokine, orchemokine is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%,99%, or 100% (w/w) of the desired oligosaccharide by weight. Purity ismeasured by any appropriate standard method, for example, by columnchromatography, thin layer chromatography, or high-performance liquidchromatography (HPLC) analysis. The nucleotides and polypeptides to bedelivered to eukaryotic cells are purified and used in delivery tohumans as well as animals, such as companion animals (dogs, cats) aswell as livestock (bovine, equine, ovine, caprine, or porcine animals,as well as poultry). “Purified” also defines a degree of sterility thatis safe for administration to a human subject, e.g., lacking infectiousor toxic agents.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a cross-sectional diagram of a delivery platform according toan example implementation.

FIG. 2 is a system block diagram illustrating a delivery systemincluding delivery platform for targeting delivery of fluid, such a dye,to tissue in vivo.

FIG. 3 is a cross sectional diagram illustrating an exampleelectro-fluidic junction.

FIG. 4 is a picture illustrating an example implementation of a deliverysystem with the example delivery platform (a) in a benchtop setup and(b) in a medical trolley.

FIG. 5 is a picture illustrating plume dispersion in the exampledelivery platform.

FIG. 6 is a table comparing properties of vectors for deliveringmolecules, such as DNA, siRNA, and proteins.

FIG. 7 includes two images showing pig trachea with methylene blue stainapplied via dripping (left) and electrospray (right), the electrosprayeddye stains the tissue, whereas dropped on dye washes off easily showingelectrospray as a viable vector for dye delivery into cells.

FIG. 8 illustrates a overlay of the spectra recorded for methylene bluebefore and after an electrospraying process, suggesting negligible or noalteration of the chemical structure of the dye.

FIG. 9 illustrates photocatalytic decomposition of methylene blue.

FIG. 10 illustrates proton nuclear magnetic resonance (1H NMR) spectraof methylene blue before and after electrospraying.

FIG. 11 illustrates proposed structures for methylene blue.

FIG. 12 illustrates staining of oral cancer with methylene blue.

FIG. 13 illustrates ionization of sulphate groups in mucins and theirsubsequent binding of methylene blue.

FIG. 14 includes pictures illustrating components of the exampleimplementation shown in FIG. 4 .

FIG. 15 illustrates electrospray delivery of plasmid DNA to explantedporcine lung tissue. (a) Representative images of green fluorescentprotein (GFP) expression following pEGFP transfection compared tonegative control (pGLuc). (b) Quantification of Gaussia luciferaseexpression following pGLuc transfection compared to negative control(pEGFP). n=21. *** p<0.001.

FIG. 16 illustrates electrospray delivery of mRNA to explanted porcinelung tissue. (a) Representative images of green fluorescent protein(GFP) expression following GFP mRNA transfection compared to buffer-onlycontrol are shown. Fluorescence image is shown as well as correspondingbrightfield image with superimposed fluorescence image, 10×magnification. (b) Quantification of luciferase expression followingluciferase mRNA transfection, compared to buffer-only control. n=9. *p<0.05.

FIG. 17 illustrates electrospray delivery of siRNA to explanted porcinelung tissue. Representative fluorescence microscopy images of FITC(model for delivery molecule) localisation following siRNA-FITC deliverycompared to buffer-only control are shown. Visualisation following (a)microdissection (b) cryo-sectioning where fluorescence image is shown aswell as corresponding brightfield image with superimposed fluorescenceimage, 20× magnification.

FIG. 18 illustrates bronchoscopic electrospray delivery of mRNA andsiRNA delivery to porcine lungs ex vivo. (a) Quantification ofluciferase expression following luciferase mRNA transfection, comparedto buffer-only control. n=3. *** P<0.001. (b) Representativefluorescence microscopy images of FITC localisation following siRNA-FITCdelivery compared to buffer-only control. Fluorescence image is shown aswell as corresponding brightfield image with superimposed fluorescenceimage, 20× magnification.

FIG. 19 illustrates an apparatus used for the electrospray delivery ofnucleic acids to porcine lung tissue segments ex vivo. (a) Electrosprayapparatus. (b) Electrospray plume, visualized with a laser, duringdelivery to tissue. (c) Magnified image of Taylor cone and electrosprayplume.

FIG. 20 illustrates bronchoscopic electrospray delivery of siRNA andmRNA to ex vivo porcine lung using an EVLP benchtop model: (a, b)electrospray apparatus; (c, d) electrospray catheter in working port ofbronchoscope; (e, f) ex vivo lung perfusion circuit including anon-invasive ventilator, peristaltic pump and temperature controlledwater bath; (g) electrospray catheter in working port of bronchoscopewith a protrusion and thermoplastic wrap for sealing and isolation.

FIG. 21 illustrates effect of electrospray solution composition onplasmid DNA nicking. (a) Solution forms droplet in the absence ofapplied voltage. (b) Typical electrospray cone-jet mode with plume whenvoltage is applied. (c) Electrosprayed DNA solutions (‘Spray’) in lowsalt, 1% ethanol or 20% ethanol were analysed by gel electrophoresis forthe incidence of supercoiled (sc) and open circle (oc) structure. Anincrease in open circle DNA was evident in the electrosprayed low saltsample. Control samples were taken before solution was placed in emitter(‘Before’), before voltage was applied to solution in emitter (‘Drip’)and from emitter after voltage removed (‘Emitter’). Representative gelfrom three independent experiments is shown.

FIG. 22 illustrates effects of flow rate and emitter size on plasmid DNAnicking. (a) Plasmid DNA was electrosprayed in low salt solution throughvarious emitter gauges at various flow rates. The ratio of supercoiled(sc) versus open circle (oc) structure was analysed by gelelectrophoresis and densitometry. Control samples were taken beforesolution was placed in emitter (‘Before’) and from emitter after voltageremoved (‘Emitter’). While there was no statistical difference betweenthe conditions, there was a trend towards the 32 gauge emitterpreserving the highest levels of supercoiled plasmid at all flow rates.(b) The voltage required to produce a stable electrospray was recordedand was found to decrease as the emitter guage increased. N=3independent experiments, ga=gauge.

FIG. 23 illustrates effect of voltage on plasmid DNA nicking. (a)Plasmid DNA was electrosprayed in low salt solution through a 32 gaugeemitter at 60 μl/min or 15 μl/min and DNA nicking was analysed by gelelectrophoresis. Increases voltage caused an increase in the incidenceof open circle (oc) structure compared with supercoiled (sc). (b)Emitter gauge did not affect the incidence of open circle structure. (c)GFP DNA was electropsrayed at varying voltages (emitter size 32 ga atflow rate 60 μl/min) and transfected with Lipofectamine 2000 into A549cells. The efficiency of GFP transfection was reduced with increasingvoltage.

FIG. 24 illustrates electrospray delivery of plasmid DNA to porcinetracheal explants. (a) Electrospray plume visualised with a laser duringdelivery to tissue demonstrating emitter, electrospray plume, tissueexplant and grounded collector. (b) Quantification of Gaussia luciferaseactivity following pGLuc transfection compared to negative control(pEGFP). n=21. *** p<0.001.

FIG. 25 illustrates electrospray delivery of RNA to porcine trachealexplants. (a) Quantification of luciferase activity following luciferasemRNA transfection compared to buffer-only control. n=9. * p<0.05.Representative fluorescence microscopy images of FITC localisationfollowing siRNA-FITC delivery compared to buffer-only control are shownfor (b) microdissected epithelial layer and (c) tracheal segmentscryosectioned in the transverse plane, 20× magnification.

FIG. 26 illustrates Bronchoscopic electrospray delivery of mRNA to wholeporcine lungs ex vivo. Quantification of luciferase activity followingluciferase mRNA transfection, compared to buffer-only control. n=3. ***P<0.001.

FIG. 27 is a table showing the effects of varying parameters such asvoltage, flow rate, needle gauge on the spray mode observed by eye.

FIG. 28 illustrates a setup to test electrospray formation using anexample porous tip of a writing instrument.

FIG. 29 illustrates a stable cone-jet mode spray formed at an exampleporous tip using 77% ethanol.

FIG. 30 illustrates electrospray formation at a porous tip using a twopower supplies.

FIG. 31 illustrates electrospray formation at an example porous tip atvarious supply voltages using 77% ethanol with a tip-to-plate distanceof 6 mm.

FIG. 32 illustrates electrospray formation at an example porous tip atvarious supply voltages using 77% ethanol with a tip-to-place distanceof 26 mm.

FIG. 33 illustrates electrospray pattern on the electrode plate obtainedby using a porous tip for no spray, tip-to-plate distance of 6 mm, andtip-to-plate distance of 26 mm, respectively.

FIG. 34 illustrates electrospray formation with 100% ethanol using anexample porous tip (standard fine brush).

FIG. 35 illustrates electrospray formation using an example porous tipwith deionized water at 4.4 kV, comparing when the electrode plate ispresent and not present. When the electrode plate is present, there is ajet ligament without a plume.

FIG. 36 illustrates electrospray formation using an example porous tipwith 0.25% methylene blue and 0.01% ammonium acetate, comparing thefoot-switch latched off and on.

FIG. 37 illustrates electrospray formation using an example porous tipwith 0.25% methylene blue and 1% ethanol.

FIG. 38 illustrates a benchtop setup to test the electrospray catheterwith an example porous tip. (a) a benchtop catheter setup, (b) close-upof the porous tip area, and (c) the catheter in a bronchoscope.

FIG. 39 illustrates (a) the electrospray catheter with an example poroustip and (b) methylene blue spray testing to ex vivo ventilated porcinelungs.

FIG. 40 illustrates the example electrospray catheter with parafilm thatcovers the metal shroud of the porous tip, tested to ex vivo ventilatedporcine lungs.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The current subject matter utilizes electrically charged spray or“electrospraying” to deliver fluid, such as dye, to tissue in vivo. Thedelivery platform can include a catheter with an electrode or chargingelement positioned within the catheter near a distal opening of thecatheter. As precise volumes of fluid meter through the catheter, theelectrode imparts charge to the fluid. When charged fluid reaches afluid/air interface at the distal opening of the catheter, a fineelectrospray of fluid is generated. Because the electrode is recessedwithin the catheter, the electrode is shielded from tissue to preventelectrical hazard to the patient and/or target tissue. Someimplementations of the current subject matter can deliver small amountof fluid (e.g., between 1 and 10 micro liters), which can cover amicroscopically visible area in vivo. Controlled delivery allows forbetter targeting of tissue for delivery, reduced trauma to tissue, andlittle or no localized pressure on tissue or enclosed area (e.g., vesselor lumen).

The term “electrospray” refers to a process where tiny, controlled andsafe quantities of electrical charge transfer to a fluid in order togenerate a very fine electrospray of the fluid. Unlike an aerosol, whicha gas mechanically and externally drives, repulsion forces internallydrive electrospray on similarly charged spray colloids. Electrospray isalso finer and significantly faster than an aerosol. In someimplementations, the device further employs a fluid-charging approach toprevent electrical hazards to the targeted tissue by shielding any andall electrodes of the delivery platform from the target tissue and/orpatient. An objective of electrospray delivery of a dye may includedifferentiating between cancer cells and normal cells in vivo such thatthe technic may be used to detect early lung cancerous changes withinthe tracheobronchial tree by staining early neoplastic epithelialchanges following electrospray.

FIG. 1 is a cross-sectional diagram of a delivery platform 100 accordingto an example implementation. The dimensions and materials shown in FIG.1 are exemplary. The delivery platform 100 includes a catheter 105having a proximal end 106 and distal end 107. The proximal end 106 anddistal end 107 are illustrated in an expanded view in FIG. 1 . Thecatheter 105 has an inner diameter defining a fluidic channel 110. Atdistal end 107 of the catheter 105 is a distal opening 115. A wire 120extends through the length of the catheter 105 and within the fluidicchannel 110. The wire 120 includes an insulation layer 122 and anelectrode 124 (e.g., an exposed portion of wire, also referred to as acharging element). The electrode 124 (e.g., exposed portion or chargingelement) is near the distal opening 115 but spaced a distance from theopening 115 to prevent direct contact between the electrode 124 andtarget tissue and/or patient thus preventing electrical hazard to thetarget tissue and/or patient. The insulation layer may be made of nylon.

A conductive sheath 125 surrounds the exterior of catheter 105 and maycouple to ground, for example, at the proximal end. The conductivesheath 125 can be grounded and can act as a shield between the electrode124 and the patient and/or tissue. The conductive sheath 125 can includea metallic braid, as illustrated in FIG. 1 .

Surrounding and enclosing at least a portion of the conductive sheath125 is a biocompatible cover 130. The biocompatible cover 130 can besmooth and lubricious for easy passage through lumens, such as abronchoscope tool channel. Biocompatible cover 130 may be formed of abiocompatible material such as, for example, polyether block amide(PEBA).

As illustrated in FIG. 1 , the biocompatible cover 130 extends less thanthe entire length of the catheter. In particular, the distal end of thecatheter 105 exposes and extends beyond the biocompatible cover 130. Itis also contemplated that the conductive sheath 125 can extend down thelength of catheter 105 further than the wire 120, but less than thecatheter 107 at the distal end of the delivery platform 100. Such aconfiguration shields the patient from electrical hazard whilepreventing grounding of charged fluid, which would hinder or preventformation of a Taylor cone. Thus, the energy used within the deliveryplatform 100 is substantially or entirely used to comminute a fluidcolumn within the delivery platform 100. There is no significant changein the energy input and output of the delivery platform 100 becauseenergy is not intended to be conducted to the body for the device tofunction.

FIG. 2 is a system block diagram illustrating a delivery system 200including delivery platform 100 for targeting delivery of fluid, such adye, to tissue in vivo. The delivery system 200 includes deliveryplatform 100, which can insert into a bronchoscope 205. The bronchoscope205 may insert into a patient's airway or other lumen during a medicalprocedure. The delivery platform 100 connects to a power supply 210 andpump 215 via an electro-fluidic junction 220. Fluid, including deliverymaterials, such as a dye, may be supplied to pump 215 from a reservoir225. A switch, such as foot switch 230, may actuate power supply 210 andpump 215 contemporaneously. FIG. 3 is a cross sectional diagramillustrating an example electro-fluidic junction 220.

Referring again to FIG. 2 , pump 215 is a metered fluid supply able tomove a precise volume of liquid in a specified time providing anaccurate flow rate. (Delivery of fluids in precise adjustable flow ratesmay be referred to as metering.) In an example implementation, pump 215may supply between 1 and 500 micro liters of fluid per actuation. Forexample, pump 215 may supply 1 to 10 micro liters or 5 to 500 microliters of fluid per actuation. In some implementations, the pump 215provides 4 microliters of fluid per actuation. Pump 215 couples(directly or indirectly) to catheter 105 so that fluid travels from pump215 to and through fluidic channel 110. In some implementations, pump215 can have a high switching time, that is, quickly reaches steadystate flow and quickly terminates flow.

Power source 210 is a high-voltage low-current supply. Power source 210can electrically couple, directly or indirectly, to the electrode 124via wire 120. The power source 210 may also provide a ground andelectrically couple, directly or indirectly, to the conductive sheath125, although, in some implementations, conductive sheath 125 may coupleto ground via another connection. In some implementations, power source210 is capable of providing between 3 and 10 kilovolts with a maximumcurrent supply of less than 25 micro amps but greater than 160 nanoamps.

In operation, the fluidic channel may be primed with fluid. Bronchoscope205 may be inserted into a patient, for example, into a patient'sairway. The delivery platform 100 may then be inserted into thebronchoscope 205 and directed towards target tissue for which a precisevolume of the fluid is to be delivered. Upon actuation of switch 230,power supply 210 can charge electrode 124 (and wire 120) to between 3and 10 kilovolts. Pump 215 can provide a metered volume of fluid to thefluidic channel 110. The electrode 124, when carrying the charge,imparts or transfers the charge to the fluid traveling through thefluidic channel 110. Due to the configuration of delivery platform 100,the charge is imparted near the distal end of catheter 105 but withinthe fluidic channel (as compared to imparting charge exterior to thefluidic channel) so that, when charged fluid reaches a fluid/airinterface near the distal opening of the catheter 105, the charged fluidforms charged droplets that disperse and form a Taylor cone.

Thus, the fluid is charged within the fluidic channel (as compared withat the fluid/air interface) and the distal opening is kept constant(e.g., the distal opening is not a nozzle, which can mechanically adjustspray characteristics, as in an atomizer or aerosol). In addition,delivery of fluid is highly localized and may be delivered to a regionapproximately 5 mm to 50 mm in diameter per actuation, limiting theexposure of fluid to non-targeted tissue and reducing collateral andunwanted effects. Moreover, delivery via electrically charged sprays hasbeen shown to possess advantageous properties over other methods, aswell as improved tissue penetration and uptake of fluids and materials,such as methylene blue dye. FIG. 6 is a table comparing properties ofvectors for delivering molecules, such as DNA, siRNA, and proteins.

In some implementations, the electrospray apparatus may include a poroustip as in FIGS. 38 and 39 within the fluidic channel 110 at the distalend 107 of the delivery platform 100. In some implementations, theporous tip may be made of an absorbent material that can retain liquidand is porous enough to allow the retained liquid to be drawn throughthe porous tip by an electric field. In certain embodiments, theporosity of the porous tip can range from about 45% to about 70% on avolumetric basis. For non-limiting examples, a felt material, a fibrousmaterial, or a filter paper formed in a cone shape can be used. Theporous tip may also be made of a soft non-woven fabric that can absorbliquid and can allow the retained liquid to be drawn through it by anelectric field.

As noted above, the porous tip can include material such as fromsynthetic fibers such as petroleum-based acrylic or acrylonitrile; orwood pulp-based rayon. Blended fibers are also possible. For example,the porous tip can be formed of one or more polymers such aspolyacrylonitrile (e.g., 85% or greater acrylonitrile monomer),polyethylene, polypropylene, polystyrene, polyvinyl chloride, syntheticrubber, phenol formaldehyde resin (or Bakelite), neoprene, nylon,silicone, and the like.

The porous tip can include fibrous material in which strands of fiberhave a diameter that can be sized to between 1 micron and 50 microns.For example, the porous tip can include fibrous material formed instrands having a diameter of about 1 micron, about 5 microns, about 10microns, about 20 microns, about 30 microns, about 40 microns or about50 microns.

Pores within the porous tip can range in size from 0.1 micron to 100microns or more. For example, the porous tip can include fibrousmaterial arranged to form pores including a diameter that are about 0.1micron, about 0.5 micro, about 1 micron, about 10 micron, about 50micron, about 75 micron, and/or about 100 micron. Other sizes arepossible.

The porous tip can be formed into a number of shapes including a coneshape. In some implementations, the porous tip can be in the shape of aTaylor cone, which can improve Taylor cone formation of an electrospray.In general, the shape of the emitter head may impact the electrosprayingmode. In some implementations, the porous tip can be sized between about0.03 mm and 3 mm in diameter. For example, the diameter can be about0.03 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about2.0 mm, about 2.5 mm, or about 3.0 mm, where about is within 10%. Thelength of the tip and hence the total fluidic resistance presented tothe sample can affect the maximum achievable flow rate for anon-continuously-fed application where fluid is drawn though the tip viathe electric field. For a continuously-fed application via syringe pumpor constant pressure supply, the length of the tip may have less impact,and fluid may be forced through the system rather than drawn out via theelectric field. The porosity of the porous tip can be selected to besufficient to facilitate the drawing of fluid through the tip via anapplied electric field.

In operation, the porous tip may absorb fluid, provide better fluidhandling, and can aid in establishing a stable cone-jet mode spraywithout a need for a pumping action. Further, as illustrated in FIG.20(g), the electrospray apparatus may include a protrusion 2001 that isdisposed near the distal opening of the catheter surrounding the outersurface of the catheter and a thermoplastic wrap 2002 that encloses theprotrusion 2001. The protrusion 2001 and the thermoplastic wrap mayprovide sealing and insulation when the electrospray catheter is used ina working port of the bronchoscope 205.

In some implementations, a porous tip can dampen, reduce and/oreliminate the effects of peristaltic forces within the fluidic channelof the electrospray catheter that can cause fluid to move through thefluidic channel. When the electrospray catheter is moved during aprocedure, peristaltic forces created by movement of the fluidic channeland connected tubing can force fluid within the fluidic channel forward,which can result in excess fluid or solution exiting the tip of thecatheter unintentionally. By including a porous tip, the porous tip canact to dampen or reduce the unintentional expulsion of fluid from theelectrospray catheter.

In some implementations, a porous tip can provide for greater controland/or flexibility over the operating parameters of an electrospraydevice. For example, adding a porous tip can allow for lower operatingvoltages while still creating a Taylor cone. Using lower voltages can beadvantageous because a payload within the delivery solution may besensitive to high voltages. Thus the porous tip can aid in maintainingthe integrity of the payload materials. As another example, utilizing aporous tip can allow for reduced flow rates while still creating aTaylor cone. Using lower flow rates can be advantageous because limitingthe amount of fluid per actuation may be clinically important forapplications where the amount of fluid delivered needs to be precise(e.g., as in the case of delivering certain chemical compounds fortherapeutic purposes). Moreover, lower flow rates can require lowerelectrical energy, e.g., voltage to form a stable spray, and lowercurrent drawn by the process, which can be beneficial from a safetyperspective. Another way to reduce the voltages can be to decrease theemitter-to-collector distance since lower distances can require lowervoltages. In addition, lower distances can produce small depositionprofiles, and thus a more targeted deposition can be achieved byshortening the distance between the emitter and collector.

In some implementations, the porous tip can be applied to electrospraydevices and platforms that are not catheters. For example, a porous tipcan be utilized within a benchtop electrospray system capable ofgeneration of particles in the micron, sub-micron and nanoparticleranges. As another example, a porous tip can be utilized for spraying asolution with a payload and permeabilizing agent (e.g., ethanol) to acellular monolayer for the intracellular delivery of the payload. Inanother example implementation, the porous tip can be utilized within amass spectrometry machine. Other examples include general surfacecoating, e.g., for printed circuit boards, and adaptive electrospraying,e.g., (Engineered Water Nano Structures) EWNS, for killing amicrobacterial organism on fruits and vegetables.

In some implementations, the porous tip can be formed with a central,axial channel forming a coaxial electrospray tip with applications indrug or cell encapsulation. The porous tip (e.g., a fibrous cone) caninclude different densities with the central channel filled with adifferent material and/or different density of fibrous material. In someimplementations, the porous tip can be utilized to electrospin, creatingfibers rather than droplets. Application of such can include for localwound healing or packing in vivo delivering drugs via core—sheath fibersIn some implementations, the porous tip can enable delivery ofelectrically conducting polymers in the form of fibers. Electrospinningsame using fibrous tip to improve consistency of fibers for use inenergy storage devices including capacitors and batteries. Otherimplementations are possible.

Although a few variations have been described in detail above, othermodifications or additions are possible. For example, the fluid mayinclude methylene blue dye (MBD). The fluid may contain DNA, siRNA,and/or proteins, which may be delivered directly to tissue. The catheter105 may be inserted into one or more of: gastrointestinal tract, oralcavity, central airway, proximal airway, small intestine, and colon.Fluid may be delivered to a portion of one or more of: bowel tissue,parathyroid gland, sentinel node, melanoma, oral tissue, and smallintestine tissue.

Other implementations are possible. By way of example and illustration,details of an example platform and uses of same are described below.

Example Implementation of Delivery Platform and System with MethyleneBlue

In an example implementation, the catheter 105 is a flexible fluorinatedethylene propylene (FEP) tube 1350 mm in length, with outer diameter1.33 mm (4 French) and an inner lumen of diameter 0.5 mm. Conductivesheath 125 is a braided shaft running the length of catheter 105terminating 50 mm short of the distal catheter tip. The inner lumen ofthe catheter 105 serves as the fluidic channel 110 and houses wire 120,which is a single strand silver wire of 0.2032 mm (AWG32) coated withperfluoroalkoxy (PFA) to a final O.D. of 0.254±0.012 mm, which forms theinsulation layer 122. The proximal end 106 of the catheter 105terminates in a Luer-lock fitting for attachment to a three-way fluidicjunction 220 to allow the combination of the PFA-coated wire 120 withpump 215. The coated silver conductor (e.g., electrode 124) terminates100 mm from the distal outlet 115 of the catheter 105 and is uncoated(e.g., exposed) over the final 2 mm to allow electrical connection withfluid.

The length of the example delivery platform (1350 mm) allowsunencumbered operation of the bronchoscope without excessive loosetubing and has an external diameter of 2.0 mm to allow facile insertioninto bronchoscope entry ports. The minimum bend radius of the exampledelivery platform is 8 mm to allow manipulation during bronchoscopyprocedures without damage, and the example delivery platform is notdamaged by bending of this severity for more than 100 cycles. Should theexample delivery platform be over-flexed and fail, any dye leak from theinner lumen is contained to prevent complications to the patient and/oroperator. The example delivery platform operates between 10° C. and 60°C. without change in performance and is not intended for use in high- orlow-pressure environments. The exterior of the example delivery platformis smooth and lubricious for easy passage via bronchoscope tool channel.All materials used in construction of this example delivery platform areapproved for use in Class IIb medical devices by the American Food andDrug Administration.

FIG. 4 is a picture illustrating an example implementation of a deliverysystem 400 with the example delivery platform. FIG. 14 are picturesillustrating components of the example implementation shown in FIG. 4 .FIG. 4(a) shows a benchtop setup for the components and FIG. 4(b) showsan exemplary implementation in a medical trolley. The medical trolleymay include lockable anti-static castors. The example delivery system400 delivers an adjustable, controlled quantity of dye via anelectrically charged spray to the lung. The example delivery system 400does not impose upon either the operator or patient a risk of electricshock during use at supply voltages of between 0 and 10 kilovolts.During use, dye should not be present at the catheter tip before thecharged spray is initiated or after it is terminated. The exampledelivery system 400 can be operated by a single user and can beactivated via a single-press foot-switch device. Delay betweendepression of the operator's foot-switch and initiation of electrosprayis minimal, and in all cases below 1 second.

The example delivery system 400 displays no hysteresis over 50electrosprays, allowing repeated doses to be delivered accurately in asingle diagnostic intervention. The example delivery platform isintended to deliver up to 50 electrosprays in a single diagnosticintervention, indwelling time lasting no more than 20 minutes. Inaccordance with ISO 10993 the example delivery platform is thereforeclassified as “limited contact (<24 hours)” or “Transient Use” inaccordance with design classification Rule 5. The example deliveryplatform is single-use and is not intended for re-sterilization or reusein multiple patients.

When the foot-switch device is actuated, the silver wire is charged tobetween 3 and 10 kV via a high-voltage low current supply, which in turncharges the dye within the catheter. The charged dye delivers via ametered pump to the catheter outlet port, creating a dye/air interfaceat the catheter outlet. The charges within the dye migrate to thisinterface and repel one another, directly countering surface tension andleading to the formation of a conical distension known as a Taylor cone.Provided the dye carries a sufficient charge this Taylor cone elutes atits apex to form a spray of charged droplets, which disassociate fromone another to form plume dispersion. FIG. 5 is a picture illustratingplume dispersion in the example delivery platform. This jetting anddispersion is used to deliver the dye to the lung, and is terminated bywithdrawing the dye into the catheter tubing by means of reversing themetered pump used in the dispense process.

The catheter component is constructed from fluorinated ethylenepropylene (FEP), which complies fully with USP Class VI and all currentISO standards, and is entirely non-cytotoxic, non-pyrogenic andnon-haemolytic. Further, FEP is shown to possess excellentparticle-shedding properties to minimize potential debris depositionwithin the lung. Voltage carrying wires within the catheter areperfluoroalkoxy (PFA)-coated Silver to maximize biocompatibility.

In the example illustrated in FIG. 4 , the following components areincluded:

-   -   1) A metered dye dispensing system capable of accurate delivery        of the dosage volumes;        -   IVEK Inc. DigiSpense 3020 Actuator and Controller; or Smiths            Medical Medfusion® 3500 Syringe Pump with PHARMGUARD® v5            Software;        -   Beckton Dickinson—Sterile disposable syringe 60 ml Plastilok            with 50 mm luer lock    -   2) An ultra low current, high voltage DC power supply for fluid        charging;        -   Spraybase/Spellman Inc.;    -   3) A footswitch to commence the delivery;        -   Schaltgeraete GmbH & Co. KG.    -   4) Medical Grade Tubing;        -   Smiths Medical Sterile Extension Set.

The following illustrates an example procedure for preparing a patient.

-   -   1. Verify the patient is a good candidate for bronchoscopy under        moderate sedation prior to initiation.    -   2. Prepare the patient for bronchoscopy. Follow patient        management protocols according to staffing, training, and        individual institution-specific policies and guidelines for        bronchoscopy.    -   3. Introduce the flexible bronchoscope through the nose or mouth        as appropriate.    -   4. Navigate the bronchoscope to the targeted site and position        the bronchoscope so that the targeted site is in bronchoscopic        view.    -   5. Prepare the airway by washing with 0.9% saline (+/−mucolytic        agents) and aspirate clear any mucus or secretions.

The following illustrates and example process for using the exampledelivery platform and system.

-   -   1. Before inserting the catheter into the bronchoscope, ensure        the protective sheath has been removed from the distal end    -   2. Advance the Catheter through the bronchoscope until the        market band on the distal tip of the Catheter shaft is in        bronchoscopic view. If the device encounters significant        resistance during insertion, do not apply excessive force. In        especially tortuous anatomy it may be necessary to relax the        bronchoscope's deflection mechanism until the device passes        smoothly.    -   3. Advance the Catheter to the targeted site under bronchoscopic        vision. Do not advance the Catheter into bronchi in which the        Catheter cannot be seen under bronchoscopic vision. Advancing        the catheter under such conditions may result in pneumothorax,        or other injury to the patient.    -   4. Do not reposition the bronchoscope with the Catheter advanced        beyond the distal end of the bronchoscope as this may result in        harm or injury to the patient.    -   5. Position the Catheter in the airway near to the target area.        Avoid touching bronchial wall with the catheter end as this may        impair Catheter function.    -   6. Deliver methylene blue to target area, by pressing and        releasing the footswitch once. The Control Module will deliver        energy automatically according to pre-set parameters for time,        voltage and current.    -   7. Once the procedure is complete and prior to manipulating the        bronchoscope, withdraw the Catheter approximately 10 cm into the        bronchoscope so the electrode array is proximal to the bend in        the distal tip of the bronchoscope.    -   8. Once the treatment is complete, remove the Catheter from the        bronchoscope. Disconnect the Catheter from the Controller, and        dispose of the used Catheter per biohazard procedures.        If mucus builds up in the airways and obscures visualisation,        remove the catheter from the bronchoscope, provide irrigation        with sterile saline, and suction the resulting fluid from the        airways. If the spray is not delivered, remove the Catheter from        the bronchoscope. Clean the distal end of the catheter with a        dry sterile swab. Confirm the Catheter has been primed and press        footswitch to visually confirm that the Catheter is functioning        properly. If it is not functioning properly, replace the        Catheter and continue with the procedure.

Experiment 1—Ex Vivo and In Vivo Large Animal

Ex Vivo

Preclinical studies were performed using an ex vivo model of freshlyexcised porcine lungs. Using the same fluid and electrical supplyequipment as the Bench Studies, 0.05% methylene blue solution wasdelivered via electrospray to various sites within the lung includingboth large and small airways. FIG. 7 includes two images showing pigtrachea with methylene blue stain applied via dripping (left) andelectrospray (right), the electrosprayed dye stains the tissue, whereasdropped on dye washes off easily showing electrospray as a viable vectorfor dye delivery into cells.

Dose responses were carried out by electrospraying variousconcentrations of methylene blue onto normal pig lungs and dissectedairway tissue. The concentration of methylene blue that gives minimalstaining when electrosprayed onto dissected pig airway tissue is0.03-0.05% methylene blue in WFI.

Criteria evaluated during animal studies were as follows; sparkformation, electrosprayed droplet size, electrical sensation/shocks tooperator, effect of bronchoscope/catheter fouling with mucus, effect ofcatheter distance from target, specificity of target area, delay ofelectrospray after foot switch actuation, repeatability of electrosprayunder identical conditions.

The primary function of the device is to comminute or atomize smallvolumes of fluid safely within the lung and airways via electrospray.Calculations to eliminate or mitigate risk were performed as well as toensure an electrospray is produced. Calculations pertaining to theelectrospray plume were confirmed by experimentation. Calculationspertaining to the device form factor were made relative to therequirement to use it with a standard video bronchoscope. In designingthe device, some calculations were confirmed by experimentation and someexperimentally obtained electrospray parameters were generalized bycalculation.

-   -   (i) Calculations of the dielectric constant of the catheter        materials to insulate the central conductor from the patient and        the operator in all circumstances.    -   (ii) Calculating the length braided earth shield over the length        of the catheter shaft. (1250 mm)    -   (iii) Calculating the minimum dead volume to determine pump        priming volume (960 uL)    -   (iv) Calculations of the ideal kV volt values to create an        electrospray from the device with the chosen fluid—normally        Methylene Blue. (4.5 kV)    -   (v) Calculation of the ideal length of the entire catheter.        (1350 mm) (vi) Calculation of minimum bend radius of catheter (8        mm)    -   (vii) Calculation of maximum width of catheter (2 mm) (˜6        French)    -   (viii) The maximum current required to produce an electrospray        was calculated/predicted using the Taylor/Melcher model (160 nA)    -   (ix) The dispense time and volume flow rate had to be calculated        to deliver the dose in the minimum time. Constraints on this        calculation included a max flow rate to maintain an optimized        spray pattern. (200 uL/min)

In-vitro tests Aims: In-vitro tests were carried out in which freshlydissected porcine airway tissue was electro sprayed ex vivo withmethylene blue. The aims of these tests were: (i) to determine theparameters under which methylene blue can be successfullyelectrosprayed; (ii) to carry out a dose response to observe thestaining pattern produced over a range of methylene blue concentrations;and (iii) to optimize a protocol for delivery of methylene blue viaelectrospray onto normal airway tissue such that minimal stainingoccurred.

Methods: Sections of airway tissue approximately 1 cm² were cut andplaced into tissue culture plates. A range of concentrations ofmethylene blue solution (0.008%, 0.015%, 0.031%, 0.04%, 0.05%, 0.063%)were made up and electrosprayed onto the dissected airway tissue.Voltages and flow rates were varied during the electrospray process inorder to determine which set of conditions were conducive to productionof stable electrosprays. The length of time that the dye was left on thetissue prior to rinsing was varied (15, 30, 60 sec). After spraying, thetissues were rinsed with a saline solution in order to observe theintensity of staining of the tissue post-spray.

Results: Parameters (voltages and flow rates) were identified at whichmethylene blue could be successfully electrosprayed in vitro onto pigairway tissue. The dose response study determined that 0.05% methyleneblue appeared to be the lowest concentration at which a stain could beobserved at the shortest incubation time of 15 sec. No staining wasobserved at the 15 sec time point with concentrations lower than 0.05%.

Conclusions: A wide range of methylene blue concentrations can besuccessfully electro sprayed by varying the voltages and flow ratesused. A solution of 0.05% methylene blue appears to be the cut off levelbelow which no staining is visible on normal airway tissue at theshortest incubation time tested here which was 15 sec. This time wasdeemed to be similar to the length of time in the clinic that it wouldtake between delivery of dye and rinsing.

Mechanical and electrical tests: An electrical measurement circuit wasestablished to perform test measurements during the design phase.Parameters tested included:

-   -   (1) Central conductor potential (4.5 kV);    -   (2) Potential (reverse) gradient within electrospray plume from        point of elution to deposition substrate/tissue (>5 kV/mm);    -   (3) Current (Ionic Current+Charge on fluid colloids) (50 nA+100        nA);    -   (4) Insulation dielectric withstand test (PFA, FEP) (>80 kV/mm);    -   (5) Tests for leakage currents and effect of guarding &        shielding (<<Noise Floor); and    -   (6) Device generated currents:        -   a. External Offset Current (<<Noise Floor);        -   b. Triboelectric Effects (<<Noise Floor);        -   c. Piezoelectric (<<Noise Floor) and Stored Charge Effects            (Energy source includes make-before-break grounding of HV            yielding Stored Charge Effects ˜0).

An electrometer (Keithley Model 6517B) was used perform a battery oftests to characterize the device. During energization, no leakagecurrent could be detected along the exterior shaft of the catheter, thefluidic or electrical connections. A mechanical bend test was performed(100 cycles) on 8 mm bend radius to visually/microscopically check formechanical wear to investigational catheter device. No wear wasobserved.

The investigational device was used in an ex-vivo porcine lung model.The device was used with an Olympus EVIS Exera BF-1T160 Bronchoscope andthe test(s) objective was to deliver 10× repeated 20 uL sprays todifferent areas in the proximal porcine airways reliably.

The data was recorded on the video card on the scope and repeatedadministration was demonstrated. The test was repeated ˜20 timesincluding on successive days.

Performance tests. Testing of prototype device was conducted using 0.05%methylene blue solution delivered via IVEK Digispense 3020 metereddelivery system, and high voltage supply was achieved via a ControlUnit. The high voltage supply may be protected with a surge protector toprevent sparks, and the Control Unit may incorporate an electrometer.The catheter device was tested for electrospray formation within anOlympus EVIS Exera BF-1T160 Bronchoscope at voltages ranging from 0 to10 kV. During testing criteria evaluated were as follows; sparkformation, electrosprayed droplet size, electrical sensation/shocks tooperator, effect of bronchoscope/catheter fouling with lubricant, effectof separation between catheter tip and electrospray target, delay ofelectrospray after foot switch actuation.

The optimal parameters for electrospray delivery of a 0.05% methyleneblue solution were 6 kV voltage, 10 μl/second delivery rate and 21 mmdistance from the device to the target area. The applied 6 kV voltagewas the minimum voltage required to enable diffuse electrospray deliveryof 10 μl methylene blue. In the tests conducted both 10 mm and 21 mmdistance was evaluated and both resulted in similar electrospray using0.05% methylene blue solution. However, 21 mm distance was determinedfrom the large animal studies as the optimal working distance fortargeted delivery of methylene blue onto the airway wall in vivo.

An evaluation of biological safety. The example delivery platform isdesigned to be entered into the lung during endoscopic procedures and isin contact with mucous membranes of the lung for <24 hours.Biocompatibility testing as recommended by ISO 10993 for limited contactdevices is therefore recommended. The catheter component is constructedfrom fluorinated ethylene propylene (FEP), which complies fully with USPClass VI and all current ISO standards, and is entirely non-cytotoxic,non-pyrogenic and non-haemolytic. Further, FEP is shown to possessexcellent particle-shedding properties to minimise potential debrisdeposition within the lung. Voltage carrying wires within the catheterare perfluoroalkoxy (PFA)-coated Silver to maximise biocompatibility.

In Vivo

Sixteen (16) normal pigs were used as an acute large animal model tosubstantiate the in vivo effectiveness of the device design. Each pigreceived 3 sprays of methylene blue with each spray lasting 1 second.

The example delivery platform was successfully used to deliver adye—methylene blue—to pig airways in a proof of principle study.Following this, in vivo experiments were performed. These experimentsinvolved anaesthetised pigs who underwent a bronchoscopy. Thisbronchoscopy allowed the investigators to inspect the surface anatomy,collect samples of bronchoalveolar fluid and electrospray methyleneblue. No adverse effects were noted in either the macroscopic airwayappearance or bronchoalveolar lavage results 4 days after theelectrospray application of methylene blue.

Rationale for selection of the model. The aim of the broad project is totranslate technology to a ‘first in man’ diagnosis of lung cancer. Forthis work, it was not possible to use large animal models of lung cancerfor several reasons. In general, the incidence of spontaneous lungcancer in animals is low. The Jaagsiekte sheep retrovirus (JSRV) that isa causative agent responsible for contagious lung tumors in sheep is notfound in Ireland. Consultation with veterinarians revealed that thelikelihood of getting permission from dog owners to use dogs with lungcancer was extremely low. Small laboratory animal models of lung cancerinduced with chemicals would not be suitable for these bronchoscopystudies. Therefore, normal pigs or sheep were options for a large animalmodel for this project to substantiate the in vivo safety andeffectiveness of the device design. Pigs were selected as the modelbecause, unlike sheep, young animals of specific size are available allyear round to ensure consistency of the model.

Study objectives. The aims of the in vivo large animal studies were to:

-   -   (i) substantiate the practicability of the device for the        clinician;    -   (ii) determine the effectiveness of the device to deliver dyes        directly to specific sites (i.e. targeted delivery) in large        animal airways in vivo;    -   (iii) select a dye that is suitable for use as a differential        stain for tumors based on the following features in normal        airways:        -   desired spreadability over the target area;        -   minimal residual staining of the target area post-rinsing;    -   (iv) devise a protocol that results in minimal staining of        non-tumour areas;    -   (v) further refine the design of the device in order to optimise        the protocol;

Conclusions:

-   -   substantiate the practicability of the device for the clinician        -   the clinicians were directly involved in all studies and            provided feedback that lead to refinement of the device and            the protocol for delivery of dye    -   (ii) determine the effectiveness of the device to deliver dyes        directly to specific sites (i.e. targeted delivery) in large        animal airways in vivo;        -   it was confirmed that the device was capable of delivering            dyes directly to specific sites within the airways of a            living and breathing large animal without encountering any            difficulties due to air movement.    -   (iii) select a dye that is suitable for use as a differential        stain for tumors based on the following features in normal        airways:        -   desired spreadability over the target area minimal residual            staining of the target area post-rinsing were confirmed    -   (iv) devise a protocol that results in minimal staining of        non-tumor areas;        -   a protocol was devised    -   (v) further refine the design of the device in order to optimize        the protocol;        -   the device was further refined in an iterative manner            throughout the series of animal studies.    -   The refinements included:        -   Removal of 2.4 mm plastic sheath at distal end extending 5            mm beyond the catheter tip since this was causing pooling of            dye between the internal radius at the sheath and the            catheter tip. The removal of the plastic sheath resolved            this problem.        -   Introduction of grounded braided shaft extending the length            of the catheter and ending 50 mm short of the catheter tip            to stiffen and protect the catheter shaft and to protect the            patient from the central conductor in an insulation fault            condition.        -   Use of a softer plastic at the catheter tip to reduce risk            of local lung trauma.        -   Introduction of a marker band 10 mm from tip to aid the            operator in locating the how far the catheter has advanced            relative to the end of the scope.        -   Reduction of the inner fluid channel lumen from 800 microns            to 500 microns in diameter. The reduced inner diameter            minimises local peristaltic flow due to bronchoscope            flexure. Such flow has not been evident or problematic.

Experiment 2—Ex Vivo Human Lung Tissue (Resected Human Lung Tumor)

A further study using ex vivo human lung cancer tissue was performed atthe Mater Misericordiae University Hospital. It concluded that adifferential methylene blue stain between normal tissue and lung cancercan be achieved. For this study, 11 patients were recruited betweenApril 2012 and July 2013. These patients had confirmed lung cancer andunderwent surgical removal of their cancer (lobectomy). Followingthoracic surgery, sections of both healthy and cancerous tissue thatwere not required for diagnosis were evaluated. This allowed theidentification of an optimal concentration range of methylene blue thatwould obtain a differential stain between lung cancer and normal tissue(when applied with an electrospray).

The concentration of methylene blue that gives optimal differentialstaining when electrosprayed onto human lung cancer tissue is 0.03-0.05%methylene blue in WFI.

Characterizing Methylene Blue in Electrospray

The electrospray technique utilized in the example delivery platform andsystem finds its first and widest application as a means of generatingions in mass spectrometry (Santos L S, A Brief Overview of theMechanisms Involved in Electrospray Mass Spectrometry, in ReactiveIntermediates: MS Investigations in Solution, 2009, Wiley-VCH VerlagGmbH & Co. and Le Gac S, Van Den Berg A, Miniaturization and MassSpectrometry, S, 2008, RCS Publishing). Electrospray Ionization (ESI) isdefined as a soft ionization technique because it can be applied toionize large molecules such as polymers, proteins, peptides and nucleicacids without fragmenting them. During the ionization process, themolecules acquire enough energy to be transferred to the gas phase butthe amount of energy remains low enough not to induce any fragmentation.

The dye methylene blue has been subjected to ESI in a number of studieswith a number of publications reporting ESI mass spectra for methyleneblue (Yang F, Xia S, Liu Z, Chen J, Lin Y, Qiu B & Chen B, Analysis ofmethylene blue and its metabolites in blood by capillaryelectrophoresis/electrospray ionization mass spectrometry,Electrophoresis 2011, 32, 659-664 and Nogueiraa F, Lopesa J H, Silvaa AC, Gonçalvesa M, Anastácioa A S, Sapagb K, Oliveira L, Reactiveadsorption of methylene blue on montmorillonite via an ESI-MS study,Applied Clay Science, 2009, 43 (2), 190-195). To verify that thechemical structure of methylene blue is not altered by the electrospraytechnology, UV-Vis spectroscopy and 1H NMR (nuclear magnetic resonance)analyses were carried out.

UV-Vis analysis. As reported in the Merck Index, methylene blue hasabsorption peaks at 668 and 609 nm. The absorbance profiles of amethylene blue sample before and after electrospraying were compared.According to Beer-Lambert's law, absorbance intensity is proportional toconcentration. If methylene blue was degraded by the electrosprayprocess, its absorption profile at 668 and 609 nm would change withrespect to the absorbance of the same methylene blue sample beforeelectrospraying.

The spectra shown in FIG. 8 demonstrate a good overlay of the spectrarecorded for methylene blue before and after the electrosprayingprocess, suggesting negligible or no alteration of the chemicalstructure of the dye. In FIG. 8 , visible light spectra for a 0.05%methylene blue solution before (−) and after (−, −) electrospraying.Electrospray setup: 3.5 cm distance emitter-collector, 6.5 kV voltageapplied to the emitter, 0.5 ml/min flow rate. The plot in the insetrepresents a magnification of the spectra.

For reference, a graph published on the open access online journalScientific Reports (Liu H, Gao N, Liao M & Fang X, Hexagonal-like Nb2O5Nanoplates-Based Photodetectors and Photocatalyst with HighPerformances, 12th January 2015, Scientific Reports 5:7716, DOI10.1038/srep07716) and showing degradation of methylene blue is shown inFIG. 9 . FIG. 9 illustrates photocatalytic decomposition of methyleneblue. The degradation of methylene blue over time is monitored by meansof UV-Vis spectroscopy. The black trace represents an untreated sampleof methylene blue. The lower absorbance intensities of the coloredtraces are due to decomposition of the dye exposed to the degradingagent Nb2O5 for different times.

FIG. 10 illustrates 1H NMR spectra of methylene blue before and afterelectrospraying. Nuclear magnetic resonance is a very sensitivetechnique to assess purity of a material as it is capable of detectingvery small amounts of impurities. In this specific case, additionalpeaks would appear if there were protons in a different chemicalenvironment due to degradation of the compound of interest. It can beobserved from FIG. 10 that the peaks for both specimens have identicalmultiplicity and chemical shift. This characteristic together with theabsence of new peaks in the sprayed sample indicate that the structureof methylene blue is preserved during the electrospray process. In FIG.10 , 1H NMR of 0.06% methylene blue and 0.06% methylene blueelectrosprayed. Electrospray setup: 3.5 cm distance emitter-collector,6.5 kV voltage applied to the emitter, 0.5 ml/min flow rate. Note: thetubing of the instrument was rinsed with acetone in order to removetraces of water resulting in a line at 2.2 ppm due to acetone.

Conclusions: The example delivery platform and system is based onelectrospray technology, which is a gentle technique to ionizematerials. Based on the data above, UV-Vis and 1H NMR spectra providedevidence that this process does not alter or degrade methylene blue.

Methylene Blue and Lung Cancer Diagnostics

Methylene blue is widely used as a dye and stain for a range of clinicalapplications. Methylene blue infusion is considered a safe and effectivemethod of localizing abnormal parathyroid glands. Methylene blue is aneffective and cheap alternative to isosulfan blue dye for sentinel lymphnode localization in patients with breast cancer. The technique ofmethylene blue staining was originally described in 1933 by Japaneseinvestigators for improving the diagnosis of early gastric cancer.Today, gastroenterology staining with methylene blue during endoscopy isa well-established method allowing a prediction between neoplastic andnon-neoplastic lesions with high specificity. Methylene blue staining isalso used for noninvasive diagnosis of melanoma and oral cancers. Lungcancer has still a very poor prognosis compared to other types of cancerand while the diagnostic value of flexible bronchoscopy for variouspulmonary diseases is well established, staining during white lightbronchoscopy (chromobronchoscopy) has not been added to the diagnosticpanel of pulmonary endoscopy up to now. Three studies have been reportedwith mixed findings regarding the efficacy of methylene blue stainingfor identification of malignant and premalignant lesions in aprospective manner. No adverse safety findings were reported and furtherresearch on chromobronchoscopy for pulmonary diseases was recommended byall three groups. The clinical uses, proposed mechanism of action andsafety of methylene blue in cancer diagnostics will be discussed below.

Vital Staining for Cancer Diagnosis

There are numerous diagnostic adjuncts available for cancer diagnosis.Cytological methods, tissue staining techniques, and molecular methodsare widely used. Supravital staining has long been used as an adjunct inthe early diagnosis of malignant lesions. In 1920's/30s, Schiller(Schiller, W. Early diagnosis of carcinoma of the cervix. Surg. Gynec.Obstet. 56 (1933). 210) first reported the use of Lugol's iodinesolution in carcinoma of the uterine cervix. In vivo staining has beenextensively used in gynaecological practice for the detection ofmalignant change of the cervix during colposcopy. The technique has beenapplied in the oral setting for over 30 years by means of the dyetoluidine blue (TB) (Kerawala C J, Beale V, Reed M, Martin I C. The roleof vital tissue staining in the marginal control of oral squamous cellcarcinoma. Int J Oral Maxillofac Surg 2000; 29:32-5). Apart from TB,other stains such as methylene blue, Lugol's iodine, and acetic acidhave also been tried in the diagnosis of cancerous lesions.

Methylene blue belongs to the family of phenothiazonium compounds. It isa cationic species and it is a basic dye (FIG. 11 ). Methylene blue iswidely used as a dye or staining agent to make certain body fluids andtissues easier to view during surgery or on an x-ray or other diagnosticexam. FIG. 11 illustrates proposed structures for methylene blue. Bothstructures shown in A and B have been proposed for MB+; however mostchemists agree that the structure of MB+ is Structure A.

Clinical Uses of Methylene Blue in Cancer Diagnostics

Barrett's Oesophagus

Barrett's oesophagus is considered to be a premalignant lesion and isfound in 10-20% of patients undergoing upper gastrointestinal (GI)endoscopy for symptoms of gastro-oesophageal reflux disease (GORD). Thenormal squamous epithelium is replaced by a specialised columnarepithelium referred to as intestinal metaplasia. Intestinal metaplasiacan evolve into adenocarcinoma in a well-definedmetaplasia-dysplasia-carcinoma sequence. Highly dysplastic or malignantBarrett's oesophagus stains differentially with methylene blue.Increased heterogeneity and decreased methylene blue stain intensity aresignificant independent predictors of high grade dysplasia and/or cancer(Canto M I, Setrakian S, Willis J E, Chak A, Petras R E, Sivak M V.Methylene blue staining of dysplastic and nondysplastic Barrett'sesophagus: an in vivo and ex vivo study. Endoscopy. 2001 May;33(5):391-400). It has been reported that a targeted biopsy is possibleto limit because methylene blue only stains non-dysplastic Barrett'smucosa but not dysplastic ones. However, many of supplementary studieshave not agreed this recommendation (Amano Y. Nihon Rinsho.Chromoendoscopic diagnosis of Barrett's esophagus. 2005 August;63(8):1416-9). For video of chromoendoscopy in Barrett's oesophagus seeDave Project 2004(http://daveproject.org/esophagus-chromoendoscopy-of-barretts-epithelium/2004-01-15/).

Bowel Cancer Surveillance

Neoplastic lesions in IBD may present as sporadic adenomas, adenoma-likemasses (ALM), dysplasia-associated lesions or masses (DALM), low- andhigh grade dysplasia's or adenocarcinomas. Chromoendoscopy is the oldestmethod used to better define the superficial gastrointestinal mucosa. Itmakes use of biocompatible colorants that are topically applied to themucosa during standard white light endoscopy through a specialized spraycatheter or water irrigation system. Different stains have been used andclassified into absorptive or contrast agents. Absorptive agents includemethylene blue (0.1-0.5%) and cresyl violet (0.2%). They enhance thesuperficial structure of lesions by exploiting the different degrees ofactive mucosal stain uptake, thus demonstrating the various cell types.Contrast agents such as indigo carmine (0.2-0.4%) highlight thearchitecture via the pooling of dye in the grooves between coloniccrypts and within the colonic pits and ridges of polyps.

Chromoendoscopy using methylene blue as a vital stain involves activemucosal absorption of the dye by small intestinal and colonic epithelium(Trivedi P J, Braden B, Indications, stains and techniques inchromoendoscopy. QMJ. DOI: http://dx.doi.org/10.1093/qjmed/hcs186 Firstpublished online: 24 Oct. 2012). The stain is not absorbed bynon-absorptive mucosa such as squamous or gastric epithelium. Targetedbiopsies should be aimed at heterogeneously stained or unstained areas,as high grade dysplasia (HGD) and early cancers absorb the dye to alower degree due to loss in goblet cells and decreased cytoplasm.

Methylene blue chromoendoscopy requires prior mucus removal from themucosal surface to ensure homogenous uptake of dye by epithelial cellsin the upper gastrointestinal tract. This can be obtained by spraying10% solution of N-acetylcysteine as a mucolytic onto the mucosal surfaceprior to the application of 0.5% methylene blue. Excess dye is carefullywashed off with water until the staining pattern is stable.

With pan-colonic dye staining, segments of 20-30 cm of colon are sprayedand evaluated at a time. A slightly lower concentration (0.1%) ofmethylene blue is applied, using a spraying catheter onto the colonicmucosa. Excessive dye is removed by suction after a staining time of ˜1min. For video of chromoendoscopy in ulcerative colitis see (DNATube).

In an international consensus statement on surveillance and managementof dysplasia in inflammatory bowel disease, (Laine, L., Kaltenbach, T.,Barkun, A., McQuaid, K., SCENIC international consensus statement onsurveillance and management of dysplasia in inflammatory bowel disease.Gastrointestinal Endoscopy. 2015; 81(3):489), the authors discussedchromoendoscopy involving the application of dye to the colon mucosa,thereby providing contrast enhancement to improve visualization ofepithelial surface detail. Methylene blue and indigo carmine were citedas the agents most commonly used, and were applied to the colon mucosavia a catheter or the colonoscope biopsy or water jet channel. pattern.Polypoid lesions were stated to be easier to detect. Once a suspiciouslesion was identified, the area was selectively sprayed with a moreconcentrated dye (indigo carmine 0.13% or methylene blue 0.2%) directlyfrom a 60-mL syringe through the biopsy channel. The consensus statementrecommended the following doses of methylene blue: 250 ml of 0.04-0.1%MB for general inspection (water jet application), with a further 30 ml0.2% MB if any suspicious lesions identified (syringe sprayapplication).

Staining Abnormal Parathyroid Glands

Methylene blue stains abnormal parathyroid glands. However,false-positive staining was reported to occur in normal parathyroidglands, lymph nodes, thyroid tissue, thymic cyst and adipose tissue.Methylene blue has been shown to be efficacious, with staining rates ofenlarged parathyroid glands approaching 100 percent (Patel H P, ChadwickD R, Harrison B J, Balasubramanian S P. Systematic review of intravenousmethylene blue in parathyroid surgery. Br J Surg. 2012 October;99(10):1345-51).

The mechanism of methylene blue staining by parathyroid glands is stillnot understood, but thiazide dyes in general (methylene blue, toluodeneblue, thionine, Azur A) are taken up preferentially in certain tissuesof the body and cleared at different rates. The staining does appear tobe somewhat related to the size of the parathyroid gland, and both chiefand oxyphil cells are readily stained, but metabolic activity or a lackof fat content or both may be factors (Orloff L A. Methylene blue andsestamibi: complementary tools for localizing parathyroids.Laryngoscope. 2001 November; 111(11 Pt 1):1901-4).

Sentinel Node Mapping

A sentinel lymph node is the first lymph node encountered by lymphaticfluid draining from a primary tumor. Intra-operative detection of thesentinel lymph node is achieved with vital dyes or lymphoscintigraphyeither alone, or in combination. The methods depend on carriage of thevital dye (Patent Blue V, Isosulfan blue or Methylene blue) and/or aradioactive nanocolloid (e.g. technetium-99m in nanocolloid) in thebreast and axillary lymphatics. The uptake kinetics of each mappingagent are different, but the function of both is to localize thesentinel node. It is thought that nanocolloids become entrapped withinthe sentinel lymph nodes either through a function of their particulatesize (the larger hydrodynamic diameter of 50-100 nm for nanocolloidrequires a transit time of usually more than 1 h) or because ofphagocytosis by leukocytes which migrate to and are retained within thedraining lymph nodes. These entrapment processes are unlikely to bemutually exclusive, and other mechanisms may also exist, but the endresult is localization of the nanocolloid within the sentinel nodesrather than its diffuse spread to secondary nodes. In contrast, patentblue dyes bind to interstitial albumin and are taken up by locallymphatic tissue. The efficiency with which the lymphatics are convertedto bright blue channels by the vital dyes reflects their smallerhydrodynamic diameter, their ability to disperse quickly and even theircapacity to readily progress through and beyond the sentinel nodes.

Melanoma

It is known that methylene blue, possesses a high affinity for melanin,a pigment present in melanoma cells (Sobal G, Rodrigues M, Sinzinger H.,Radioiodinated Methylene Blue—A Promising Agent for MelanomaScintigraphy: Labelling, Stability and In Vitro Uptake by MelanomaCells. ANTICANCER RESEARCH 28: 3691-3696 (2008)). Methylene blue forms astrong complex with melanin and may provide a means of selectivedelivery of radionuclides to melanoma cells, useful for noninvasivediagnosis as well as for therapy of disseminated disease. The fact thatmethylene blue is not directly toxic to the tumor and accumulates inmelanoma tissue, showing a high concentration of melanin, allows tumorimaging using suitable radionuclides.

Oral Cancer

Most oral malignancies occur as squamous cell carcinomas. Many OSCCsdevelop from premalignant lesions & conditions of the oral cavity. Theexact mechanism for the uptake of methylene blue in epithelial tissuemay resemble that of toluidine blue in the acidophilic characteristic ofcells with abnormal concentration of nucleic acid, resulting indifferential uptake between normal/benign and highlydysplastic/malignant cells.

Periodic clinical examination of the oral cavity is the mainstay forearly detection of oral cancers. It was shown to reduce mortality fromoral cancer by 32% in high-risk individuals. Additionally, usingadjunctive aids such as toluidine blue (also referred to as toloniumchloride) has been widely accepted to improve the effectiveness inlarge-scale screening for oral cancer diagnosis. However, it ishazardous if swallowed, and was shown to have toxicity to fibroblasts.Methylene blue has a similar chemical structure and exhibits similarphysicochemical properties to toluidine blue but is less toxic to thehuman body. The sensitivity and reliability of in vivo staining withmethylene blue as a diagnostic adjunct in screening for oral malignantor precancerous lesions methylene blue has been evaluated and methyleneblue has been shown to be a useful diagnostic adjunct in a large,community-based oral cancer screening program for high-risk individuals(Ya-Wei Chen, Jiun-Sheng Lin, Cheng-Hsien Wu, Man-Tien Lui, Shou-YenKao, Yao Fong. Application of In Vivo Stain of Methylene Blue as aDiagnostic Aid in the Early Detection and Screening of Oral SquamousCell Carcinoma and Precancer Lesions. J Chin Med Assoc, 2007.70:497-503).

For the procedure, the mucosa of the target area is gently dried withgauze and power air spray to ensure that the lesion is not beingcontaminated with saliva. The dye (1% methylene blue) is directlyapplied on the lesion with help of cotton bud first and after used as amouth rinse. Patients gargle with 1% Methylene blue for 30 seconds; thenexpectorate. Patients then rinse again with 1% lactic acid for 30seconds to wash out the excess dye. The pattern of dye retention isassessed by the intensity of stain retention on the lesion (FIG. 12 )(Riaz A, Shreedhar B, Kamboj M and Natarajan S. Methylene blue as anearly diagnostic marker for oral precancer and cancer. SpringerPlus2013, 2:95 doi:10.1186/2193-1801-2-95).

Lung Cancer

There have been 3 published reports of the utility of methylene blue asa potential lung cancer diagnostic when used during bronchoscopy. Theresults of these were conflicting, with the 2 case series from the 1980sreporting a potential benefit and a more recent study that was unable tovalidate these previous results.

-   Chromobronchoscopy in the Diagnosis of Brochial Tumours (Ovvhinnikov    A A, Dianov V V, Lukomosky G I. Chromobronchoscopy in the Diagnosis    of Bronchial Tumours. Endoscopy. 1980; 12:147-150)

Protocol: Rigid bronchoscopy under general anaesthetic using a Friedelbronchoscope. The bronchial area to be stained were washed twice with 5%sodium carbonate (or physiological saline) and unspecified volume oftrypsin or chymotrypsin. 2 ml of 0.2-0.4% methylene blue solution wasdelivered to the target area using a bronchoscopic atomiser. After 1minute, the area was washed with physiological saline. Photographs andbiopsies of the stained areas were then taken.

Results: 140 patients were included in the study. 76 patients werediagnosed with non-small cell lung cancer (NSCLC) based on biopsyresults. In all 76 lung cancer patients, methylene blue was reported toselectively stain lung cancer. The stain is described as “dark blue”with “sharp contrast to the pink mucosa of unaffected areas”. Squamouscell carcinomas were described as staining more intensely than otherforms of NSCLC. Benign bronchial tumours were not stained. 16/60patients without cancer were stained by methylene blue, however this wasdescribed as “less intense as lung cancer”. These false positive resultsoccurred in patient with infective or inflammation disease (biopsyresults showed metaplastic change in 10/16 of these patients).

-   Vital Staining in Fibre optic Bronchoscopy (Varoli F, Mariani C,    Fasceanella A, Cosentino F, Vital Staining in Fibreoptic    Bronchoscopy. Endoscopy. 1986; 18:142-143.)

Protocol: Flexible bronchoscopy under awake sedation. Target area of thebronchial tree were washed with mucolytic solution (ambroxol chloride),5% sadium carbonate and physiological saline. An unspecified volume of0.7% methylene blue or 0.5% toluidine blue was applied to the targetarea using a spray catheter. After 1 minute, the target area was washedwith physiological saline. Biopsies were taken from visually identifiedexophytic tumours, whether stained or not, as well as areas of stainingin visually normal mucosa.

Results: 28 patients were included in the study. Methylene blue wasutilised in 18 cases. Lung cancer was visually identified duringbronchoscopy in 21 patients, and confirmed by biopsy. Selective stainingof lung cancer was reported in 17/21 cases. In 6 of these patients,further areas of “minor” staining were noted in visually normal regions.Biopsy reveal squamous metaplasia (5/6 patients) and severe dysplasia.Of the 7/28 patients without exophitic tumour growth, 4 patients showedpositive staining. Biopsy of these areas revealed epideroidal carcinoma(1/4) and squamous metaplasia (3/4). Staining of the areas of squamousmetaplasia was described as “less intense than neoplastic areas”.

-   Methylene Blue-Aided In Vivo Staining of Central Airways during    Flexible Bronchoscopy (Zirlik S, Hildner K M, Neurath M F, Fuchs    F S. Methylene blue-aided in vivo staining of central airways during    flexible bronchoscopy. Scientific World Journal. 2012; 2012:625867.    doi:10.1100/2012/625867)

Protocol: Flexible bronchoscopy under awake sedation. Thetracheobronchial tree was examined using white light bronchoscopy toidentify macroscopic areas of abnormal mucosa. Prior to the applicationof methylene blue, the airways were prepared by applying 5 ml of 5%acetylcysteine (mucolytic), 10 ml of 0.9% sodium chloride and aspiratingsecretions. 10 ml of 0.1% methylene blue was then applied using a spraycatheter. After 1 minute, the target area was washed with physiologicalsaline. Biopsies were taken from areas of circumscribed or remarkabledye uptake, as well as from areas identified as possibly malignant onwhite light bronchoscopy.

Results: 26 patients were included in this study. In all patients aweak, non-specific staining of the whole bronchial tree was noted. In 11patients with exophytic lung cancer visible during white lightbronchoscopy, no preferential methylene blue stain was reported. Thisstudy reported that significant uptake of methylene blue was only seenin 1 of 19 patients diagnosed with cancer on lung biopsy. In 1 case,normal mucosa was stained blue but the lung cancer remained unstained.No side effects of the staining procedure occurred during bronchoscopyor during a follow up of 24 hours in all patients.

Methylene Blue Vital Staining: Proposed Mechanisms of Action

Methylene blue is used as a contrast stain in several diagnosticsituations. In some tissues, methylene blue stains the normal epitheliumbut does not stain metaplastic/cancerous cells while in other tissues,methylene blue does not stain the normal epithelium and does stainstains metaplastic/cancerous cells (Fennerty M B, Sampliner R E, McGee DL, Hixon L J, Garewal H S. Intestinal metaplasia of the stomachidentification by a selective mucosal staining technique. GastrointestEndosc 1992; 38:696-698 and Fennerty M B. Tissue staining. GastrointestEndosc Clin North Am 1994; 4:297-311). For example, methylene blue istaken up by actively absorbing tissues such as small intestinal andcolonic epithelium. It has been used to highlight subtle mucosal changesin the small intestine (e.g. celiac disease) and colon (flat adenomasand carcinomas). It does not stain non-absorptive epithelia such assquamous or gastric mucosa. Hence, it can positively stain metaplasticabsorptive epithelium, such as intestinal-type metaplasia in the stomachor not stain non-absorptive epithelium, such as ectopic gastricmetaplasia in a background of positive staining duodenal mucosa.

Thus, methylene blue staining of normal epithelium is due to normalactive absorbing processes in certain tissues and a contrast is apparentwith metaplastic/cancerous cells, which appear unstained in comparison.In tissues that are not solely absorbing tissues, such as the oral andairway epithelia, methylene blue does not stain the normal epithelium.The mechanism by which methylene blue stains metaplastic/cancerous cellsin these tissues is not fully understood. Methylene blue can enter cellswhen there are structural alterations in the cell membrane which mayoccur in cancer cells. Alternatively, the mechanism for the uptake ofmethylene blue in epithelial cells may resemble that of toluidine bluein the acidophilic characteristic of cells with abnormally increasedconcentrations of nucleic acids, resulting in differential uptakebetween normal/benign and highly dysplastic/malignant cells. Chen andco-workers published a study in 2006 where they assessed the use ofmethylene blue as a diagnostic aid in early detection of oral cancer andprecancerous lesions (Chen et al. Use of methylene blue as a diagnosticaid in early detection of oral cancer and precancerous lesions. BritishJournal of Oral and Maxillofacial Surgery Volume 45, Issue 7, October2007, Pages 590-591). The sensitivity was comparable with that reportedusing toluidine blue staining. Cationic dyes are commonly called basicdyes and so substances staining with such dyes are called basophilic.Substances that bind basic dyes include nucleic acids and acid mucins.Positively charged methylene blue ions will bind to tissue anions suchas carboxylic acids, sulphuric acid and phosphoric acid groups. Thesegroups need to be ionised to bind to the dyes (FIG. 13 ).

Safety

Acute Toxic Effects:

-   -   1. In humans, large doses (500 mg) administered intravenously        have been reported to cause nausea, abdominal and chest pain,        cyanosis, methemoglobinemia, sweating, dizziness, headache and        confusion.    -   2. Intradermal and subdermal injections of Methylene blue can        cause erythematous skin lesions, superficial ulceration and        tissue necrosis.    -   3. 3 reported anaphylaxis reactions to methylene blue:        -   2005: Intrauterine 1% methylene blue instillation        -   2008: SLN mapping in breast.        -   2010: SLN mapping in breast, 2 ml subdermal methylene blue

Safety Data:

-   -   1. Safer than Isosulfan (Lympazurin)        -   Bezu C et al., Surg Oncology, 2011.        -   In SLN mapping for gastrointestinal tumours. Soni M et al.,            Ann Surg Oncol, 2009.    -   2. Expected to be safe for use in pregnancy, minimal fetal risk        -   Pruthi S et al., Amer J Surg, 2011.    -   3. methylene blue is safe for use in children to distinguish        between preauricular sinuses (PASs) and branchial sinuses and        fistulae (BSF).        -   Dickson J M et al., J Otolaryngol Head Neck Surg, 2009.

Adverse Device Effects

Techniques for early diagnosis of diseases such as lung cancer areurgently required. Lung cancer remains the largest cause of cancerdeaths worldwide. Lung cancer is accountable for the highest number ofcancer deaths worldwide, despite advances in chemotherapy over recentyears. The most effective method to substantially improve survivalfigures would be diagnosing lung cancer at an earlier stage, whentherapy may be more effective. The current electrospray platform mayallow for a novel diagnostic test that would improve the accuracy ofinvasive tests (bronchial bisopsies).

The standard risks associated with bronchoscopy are rare and includeexcessive bleeding from a biopsy site, low oxygen levels due to thesedation and heart arrhythmias. Very occasionally a patient may complainof a sore throat or a fever after the procedure.

The risks of the use of small volumes of topical methylene blue to thelungs is felt unlikely to impart any significant side effects, and thishas already been applied to the lung at a dose of an unspecified volumeof 0.7% methylene blue using a spray catheter over a period of 1 minutewithout adverse events reported (Zirlik S, Hildner K M, Neurath M F,Fuchs F S. Methylene blue-aided in vivo staining of central airwaysduring flexible bronchoscopy. Scientific World Journal. 2012;2012:625867. doi:10.1100/2012/625867). In addition, intravenousadministration of methylene blue is routinely used in the treatment ofmethemoglobinaeimia and as a visual aid in parathyroid surgery.

In regards to the risk of bronchoscopic electrospray, this is a novelprocedure that has not as yet been performed in man. As such the adverseevents are unknown. The platform is a low energy system that is notthought to be associated with risk to the patient. However, to atomisethe methylene blue an electrical current is applied to solution. Thepotential risks associated with this include device failure, electricshocks, burning to lung tissue and cardiac arrhythmias.

The use of small volumes of topical methylene blue delivered in thisstudy are unlikely to impart any significant side effects, the reportedside effects of the use intravenous of methylene blue include thefollowing:

-   -   The most commonly reported adverse reactions are nausea,        abdominal and chest pain, headache, dizziness, tremors, anxiety,        confusional state, dyspnoea, tachycardia, hypertension, the        formation of methaemoglobinaemia and hyperhidrosis.    -   It may impart a blue-green color to urine and a blue color to        skin    -   Repeated doses of methylthionium chloride may exacerbate Heinz        body formation and haemolytic anaemia (Total cumulative dose        should not exceed 4 mg/kg)    -   Methylthionium chloride should be avoided in patients receiving        medicinal products that enhance serotonergic transmission        including SSRIs (selective serotonin reuptake inhibitors),        bupropion, buspirone, clomipramine, mirtazipine and venlafaxine.        Example Implementation of Electrospray-Mediated Transfer of        Nucleic Acids to Ex Vivo Porcine Lung

While nucleic acid-based therapies have the potential to provideclinically meaningful benefit across a wide spectrum of lung disease invivo delivery remains a challenge. Electrospray atomisation is a methodof generating fine droplets of a solution. Here we examined thefeasibility of using electrospray to deliver nucleic acids to explantedporcine lung tissue and whole lungs. Reporter plasmid DNA and mRNA,expressing green fluorescent protein and luciferase, and FITC-labelledsiRNA were delivered in 20% ethanol solution via electrospray toexplants of tracheal tissue cultured at air-liquid interface. Increasedfluorescence was evident in explants following delivery of pEGFP, GFPmRNA and siRNA-FITC compared to controls. Luciferase levels in explantculture supernatants following electrospray transfection of pGLuc(p<0.005) and luciferase mRNA (p<0.05) were significantly increasedcompared to controls. Solutions of luciferase mRNA and siRNA-FITC werealso delivered to a porcine ex vivo whole lung model via bronchoscopyusing an electrospray catheter. Successful bronchoscopic electrospraydelivery of luciferase mRNA (p<0.005) and siRNA-FITC was observed. Insummary, we report the successful ex vivo delivery of nucleic acids toexplanted porcine lung tissue via electrospray. In addition, we describebronchoscopic electrospray delivery of nucleic acid to ex vivo porcinewhole lung.

Nucleic acid-based therapies have the potential to provide clinicallymeaningful benefit across a wide spectrum of lung disease. However,challenges in achieving effective delivery have limited the success ofthis therapy to date. While viral vectors have been intensively examinedfor lung gene therapy, the airway mucus gel layer remains a significantbarrier to inhaled viral and non-viral vectors¹. Non-viral methods ofgene delivery, including chemical and physical methods of gene delivery,have benefits over viral delivery as they are less immunogenic, lessrestricted in the size of DNA they can deliver and have fewer regulatoryhurdles for in vivo use. However, the absence of a successful commercialtherapy involving non-viral vectors indicates that further challengesremain with these approaches.

In addition to delivery of traditional drugs, aerosolisation has longbeen viewed as a desirable route for gene delivery to the lungs.Aerosolization of a solution can be achieved by several methods. Themost common method employs a spray nozzle through which fluid passes andis acted upon by mechanical forces that atomize the liquid. For deliveryto the lung, sonication is widely used whereby high frequent vibrationthrough a nebulizer nozzle plate produce small droplets of a liquid.However, current aerosol delivery methods such as jet or ultrasonicnebulisers can cause shear stress to the DNA resulting in a poorefficiency of gene delivery², a significant drawback of devices andmethods prior to the present invention overcomes the drawbacks ofearlier methods and devices. Electrospray, also known as electrostaticspray, is an alternative method of atomizing a liquid. In order togenerate an electrospray, a high voltage is applied to a solution as itpasses through a conducting emitter such as a needle³. The potentialdifference generated between the charged solution and a ground electrodegenerates an electric field drawing the liquid towards the groundelectrode. The meniscus forms a characteristic cone, known as a ‘Taylor’cone and when the voltage threshold overcomes the surface tension of thesolution, the tip of the Taylor cone dissociates, or atomizes, intodroplets forming either a jet, a plume or a combination spray of chargedmicrodroplets. With other methods of spray generation the droplets fallby gravity onto a surface but with electrospray, within a certaindistance, the droplets accelerate towards a surface⁴. In addition,because the droplets are charged, an electrospray is more controllablethan other forms of spray, yet another advantage over earlier attempts.Unlike previous attempts, transfection of delivered molecules andfunction of those molecules in pulmonary cells was demonstrated atair-liquid interface using the device and methods described herein.

DNA, mRNA and siRNA via electrospray were delivered to lung tissue at aclinically-relevant air-exposed interface, e.g., to porcine trachealexplants cultured at an air-liquid interface. Then, we assessed thefeasibility of electrospray atomisation for the delivery of nucleicacids directly to the lung, via bronchoscopy, in a porcine model.

Materials and Methods

Materials: Plasmids: pEGFP (Clontech Laboratories, Mountainview, Calif.,USA) and pGLuc (New England Biolabs, Ipswich, Mass., USA). GFP mRNA andluciferase mRNA (TriLink Biotechnologies, San Diego, Calif., USA).BLOCK-iT™ Fluorescent Oligo (siRNA-FITC) (Thermo Fischer Scientific,Waltham, Mass., USA).

Electrospray Delivery

The Spraybase Generation 1 electrospray instrument with a 30G Hamiltonneedle was used (Avectas Ltd., Maynooth, Ireland) (FIG. 19 ). Theemitter, a, was positioned above a grounded base plate, 15 mm from theepithelial surface of the tissue. Voltage was adjusted to achieve astable Taylor cone and diffuse plume (FIGS. 19 b and 19 c ). The voltagerequired to achieve this ranged from 3-5 kV depending on environmentalconditions (humidity/temperature), flow rate and concentration ofsolution. Nucleic acids were re-suspended in delivery solution which waswater containing 20% ethanol. Porcine explants were placed epithelialsurface up, in the center of the base plate, below the electrosprayemitter and nucleic acid solutions were delivered over 2-3 minutes, at aflow rate appropriate for the solution. For experiments involvingplasmid DNA, the total treatment (15 μg) was delivered in three divideddoses, on three consecutive days. In all other experiments the totaltreatment was administered with a single spray. Following nucleic aciddelivery, the tissue segments were placed on agarose plugs in freshculture medium.

Bronchoscopic Electrospray Delivery of Nucleic Acids to Porcine Lung ExVivo

The electrospray catheter system was composed of an electrospraycatheter, power pack and a syringe pump (Avectas Ltd.) (FIG. 20 ). Theporcine heart-lung block was prepared by cannulating the superior venacava and perfusing culture medium supplemented with 7% albumin and 0.5%dextran through the pulmonary vasculature with a peristaltic pump(Masterflex, Gelsenkirchen, Germany). The trachea was intubated andventilated (IPAP 14; EPAP 4; FiO2 21%) using a NIPPY 2 ventilator(RespiCare Ltd., Swords, Ireland). The lungs were cleared of debris byinstilling 10 ml aliquots of 0.9% saline and suctioning. Prior tobronchoscopic electrospray delivery, a target endobronchial area wasselected and marked with 3 dots to form a target using SPOT endoscopicmarker (GI Supply, Camp Hill, Pa., USA).

Analysis of Nucleic Acid Expression

Fluorescence was analysed using the Olympus CKX41 microscope (OptikaVision Pro) and fluorescence module (CoolLED pE-300white). Some GFP mRNAand siRNA-FITC electrosprayed tissues and controls were frozen in OCT(Sigma-Aldrich) and cryosectioned prior to viewing by fluorescencemicroscopy. For pGLuc and luciferase mRNA expression, followingappropriate incubation periods, supernatant was analysed using theBiolux™ Gaussia Luciferase Assay (New England Biolabs) according tomanufacturer's instructions.

Statistics

Two-tailed, unpaired T-Tests were used for statistical analysis usingGraphpad Prism version 7.03 for Windows (GraphPad Software, La Jolla,Calif., USA).

Results

Delivery of Plasmid DNA to Lung Explant Tissue

Porcine tracheal explants were electrosprayed with 5000 ng of pEGFPplasmid DNA (833 ng/μl; flow rate 3 μl/min; duration 2 min) on day 1, 2and 3 and then cultured for a further 48 hr. pGLuc, which encodes forluciferase, was delivered as a negative control for green fluorescentprotein (GFP) fluorescence. The epithelial and submucosal sections werethen dissected from the trachea and underlying connective tissue.Sections were mounted on slides and visualized by fluorescencemicroscopy. GFP expression was evident in the tissue sections ofpeGFP-treated tissue but not in the pGLuc negative control (FIG. 15 a ).

It was noted that high levels of endogenous auto-fluorescence present inlung tissue can impede visualization of fluorescent reporters.Therefore, pGLuc was used to further validate and quantify DNAexpression after electrospray delivery. 5000 ng pGLuc was electrosprayedonto tracheal tissue sections (833 ng/μl; flow rate 3 μl/min; duration 2min) for 3 consecutive days and tissues were then cultured for a further48 hours. peGFP was delivered as a negative control. 20 μl of culturemedia was sampled from each well and luciferase expression wasquantified. There was a significant increase of luciferase expression(p=0.0002) in the media of pGLuc electrosprayed tissue compared to themedia of pEGFP electrosprayed controls (FIG. 15 b ).

Delivery of mRNA to Lung Explant Tissue

We next examined the delivery of mRNA encoding for GFP and luciferase.2.4 μg GFP mRNA was electrosprayed onto tracheal explants (400 ng/μl;flow rate 3 μl/min; duration 2 min) and cultured for 24 hr. Bufferwithout mRNA was electrosprayed as a negative control. GFP expressionwas evident in the dissected epithelial layer of the tracheal tissuewhen examined by fluorescence microscopy (FIG. 16 a ). 10 μg luciferasemRNA was electrosprayed onto the trachea explants (700 ng/μl; flow rate4.8 μl/min; duration 3 min) and incubated for 48 hours. Again bufferwithout mRNA was electrosprayed as a negative control. Quantification ofluciferase expression showed a significant increase in luminescenceexpression (p=0.023) in the mRNA electrosprayed samples compared tocontrol explants (FIG. 16 b ).

Delivery of siRNA to Lung Explant Tissue

We were also interested in the ability of electrospray to achievedelivery of siRNA molecules. 20 μl 100 μM siRNA-FITC was delivered tothe trachea explant (100 uM; flow rate 10 ul/min; duration 2 min) andcompared to a buffer-only negative control. Following delivery, theepithelial layer was microdissected and examined under a fluorescentmicroscope. FITC fluorescence was evident in tissues that wereelectrosprayed with siRNA but not in control tissues (FIG. 17 a ). Inaddition, tracheal segments were cryosectioned in the transverse plane,allowing visualisation of siRNA within the tissue (FIG. 17 b ).

Bronchoscopic Delivery of mRNA and siRNA to Porcine Lung Ex Vivo

Having demonstrated successful delivery of nucleic acids to explantedlung tissue, we next examined whether we could achieveelectrospray-mediated delivery into a whole lung using a bronchoscopeand an electrospray catheter. Prior to bronchoscopic electrospraydelivery, a target endobronchial area was selected and marked with 3dots to form a target using SPOT endoscopic marker. The electrospraycatheter was then inserted into the working port of a bronchoscope,positioned 15 mm from the pre-marked target and nucleic acid wasdelivered to the center of the dots. Six 20 μl aliquots of siRNA-FITC(3.3 μM solution; flow rate 100 μl/sec; duration 0.2 sec per aliquot)and 10 μl (3 μg) of mRNA-GLuc (300 ng/μl; flow rate 100 μl/sec; duration0.1 sec) were delivered to target areas. These were then resected,washed as above and cultured at air liquid interface. Luciferase mRNAexpression was measured at 48 hr post-delivery and siRNA-FITC was viewedat 24 hrs post-delivery. A significant (p<0.0001) increase in luciferaseexpression was detected in luciferase mRNA-treated samples compared tobuffer-only controls and visible fluorescence following siRNA-FITCdelivery was apparent (FIG. 18 ).

Porcine Tracheal Explant Culture

Pig lungs were obtained from a local abattoir. Cold ischemic time waslimited to 90 min. The trachea was dissected into sections approximately20×50 mm, rinsed with phosphate-buffered saline (PBS) and 3-4 washcycles in 1:1 RPMI 1640:DMEM, 200 units/ml penicillin, 200 ug/mlstreptomycin, 2.5 ug/ml amphotericin, 50 ug/ml gentamicin (allSigma-Aldrich, St. Louis, Mo., USA) were performed. Each wash cycleincluded changing wash media, 10 minutes of agitation on a cell shakerand 1 hr incubation at 37° C., 5% CO₂. Tracheas were cut into 10 mm²segments, including epithelium, mucosa and cartilage. Tissue segments,epithelial layer facing upwards, were placed onto 5 mm high sterileagarose (1% w/v) plugs in 12-well plates. Culture media (wash media plus10 mM L-glutamine and 10% FBS (Sigma-Aldrich)) was added so that thebasal section of the tissue section was submerged. Explants wereincubated overnight at 37° C., 5% CO₂, in a humidified atmosphere.

Nucleic Acid-Based Therapies Using Electrospray Catheters

Nucleic acid based therapies have significant disease modifyingpotential. As such, there has been much interest in developingtechniques that successfully deliver these molecules in vivo. The datadescribed herein was generated using art-recognized models for humanclinical or veterinary clinical therapeutic administration modalities.Multiple viral and non-viral vectors have been utilized for this aim,but success has been limited to date. Challenges of cytotoxicity,immunogenicity and transfection inefficiency have hampered progress.Improved safety profiles with non-viral vectors have increased interestin these techniques. However, technologies to improve transfectionefficacy are needed if non-viral vector therapies are to have aclinically meaningful effect. In this study, we have shown thatvector-free delivery of nucleic acids to tissue using electrospray isnot only feasible but efficiently delivered, thereby reducing cost.Moreover the delivered molecules are functional, and the mode ofdelivery is minimally invasive to the subject being treated.Furthermore, using the electrospray catheter described herein, atechnique was developed that allows for luminal delivery, e.g.,bronchoscopic lung delivery, demonstrating the efficacy of thistechnology in the clinic.

This study reports the successful electrospray delivery of RNA, DNA,proteins or other molecules to tissue. Porcine lung was selected as atarget for gene delivery in view of its extensive use in translationalresearch and its close approximation of the human lung for bronchoscopicintervention^(7,8). Furthermore, the epithelial surface of the lungprovides an accessible target for non-invasive (inhalation) andsemi-invasive (bronchoscopy) drug delivery. Electrospray atomisation hasthe potential to be utilized as a drug delivery platform for bothmodalities.

The process of electrospray atomisation involves the generation of anelectric field between a charged solution and a grounded collector. Asthe electromagnetic field overcomes surface tension, the solution isdispersed into nano-sized particles that move towards a collector athigh velocity. The characteristics of electrospray generatedparticles—size, charge, velocity and direction—facilitates drug deliveryby addressing factors that reduce the efficiency of nucleic aciddelivery to tissue. Electrospray creates conditions in which particlespass through a cell membrane by kinetic force. This effect may also beachieved by the interaction of charged particles with membrane-boundvoltage gates or ion channels. In addition, the electric fieldassociated with an electrospray may induce the transient formation ofpores within the cell membrane, known as electroporation.

Electrospray ionisation is a vector-free delivery system that has thepotential to overcome multiple barriers associated with inefficienttransfection. This technique has previously been reported tosuccessfully transfect plasmid DNA in vitro.^(12,13) Here we report thesuccessful electrospray delivery of nucleic acids, e.g., plasmid DNA,mRNA and siRNA molecules. Protein expression following transfection wasidentified, indicating that biological integrity of nucleic acidsolutions are maintained during the electrospray process.

Delivery of plasmid DNA to tissue can be challenging because the largesize of these molecules can negatively impact upon cellular uptake. Inaddition, cellular uptake of DNA does not guarantee protein expressionas nucleolar relocation must occur before protein encoding can commence.Delivery of smaller molecules, such as mRNA, increases the efficacy ofprotein expression by removing barriers associated with molecular sizeand ribosomal transcription. mRNA may also have therapeutic advantagesover DNA, where factors such as transient protein expression may be animportant consideration in drug development. A 2 Log increase inluciferase expression was seen with both plasmid DNA and mRNA comparedto controls.

Gene regulation, and therefore disease modulation, by siRNA moleculesalso have great potential for treating conditions such as cancer.¹⁴ Wehave shown that fluorescent labelled siRNA molecules were be deliveredto the epithelial and mucosal layers of lung tissue by electrospray.Delivery of siRNA function is useful to downregulate disease pathwayssuch as infection, inflammation or oncogenesis.

REFERENCES

-   1. Duncan G A, Jung J, Hanes J, Suk J S. The Mucus Barrier to    Inhaled Gene Therapy. Mol Ther. 2016. 24(12):2043-2053.-   2. Gomes Dos Reis L, Svolos M, Hartwig B, Windhab N, Young P M,    Traini D. Inhaled gene delivery: a formulation and delivery    approach. Expert Opin Drug Deliv. 2017. 14(3):319-330.-   3. Jaworek A., Sobczyk A. T. Electrospraying route to    nanotechnology: An overview. Journal of Electrostatics. 2008.    66:197-219.-   4. Rosell-Llompart, J. & Fernández de la Mora, J. Generation of    monodisperse droplets 0.3 to 4 mm in diameter from electrified    cone-jets of highly conducting and viscous liquids, J. Aerosol    Sci., 1994. 25, 1093-1119.-   5. Okubo Y., Ikemoto K., Koike K., Tsutsui C., Sakata I., Takei O.,    Adachi A., Sakai T. DNA Introduction into Living Cells by Water    Droplet Impact with an Electrospray Process. Angew. Chem. Int. Ed.    2008, 47, 1429-1431.-   6. Zeles-Hahn M G., Lentz Y K., Anchordoquy T J., Lengsfeld C S.    Effect of electrostatic spray on human pulmonary epithelial cells.    Journal of Electrostatics. 2011. 69:67-77.-   7. Rogers C S, Abraham W M, Brogden K a, et al. The porcine lung as    a potential model for cystic fibrosis. Am J Physiol Lung Cell Mol    Physiol 2008; 295: L240-63.-   8. Judge E P, Hughes J M L, Egan J J, Maguire M, Molloy E L,    O'Dea S. Anatomy and bronchoscopy of the porcine lung: A model for    translational respiratory medicine. Am. J. Respir. Cell Mol. Biol.    2014; 51: 334-43-   9. Zhang D, Das D B, Rielly C D. Potential of microneedle-assisted    micro-particle delivery by gene guns: a review. Drug Deliv 2013;    7544: 571-87.-   10. Lambricht L, Lopes A, Kos S, Sersa G, Préat V, Vandermeulen G.    Clinical potential of electroporation for gene therapy and DNA    vaccine delivery. Expert Opin Drug Deliv 2015; 5247: 295-310.-   12. Ikemoto K, Sakata I, Sakai T. Collision of millimeter droplets    induces DNA and protein transfection into cells. Sci Rep 2012; 2:    1-5.-   13. Lee Y H, Wu B, Zhuang W Q, Chen D R, Tang Y J. Nanoparticles    facilitate gene delivery to microorganisms via an electrospray    process. J Microbiol Methods 2011; 84: 228-33.-   14. Golan T, Khvalevsky E Z, Hubert A, et al. RNAi therapy targeting    KRAS in combination with chemotherapy for locally advanced    pancreatic cancer patients. Oncotarget 2015; 6: 24560-70.-   15. Zamora M R, Budev M, Rolfe M, et al. RNA interference therapy in    lung transplant patients infected with respiratory syncytial virus.    Am J Respir Crit Care Med 2011; 183: 531-8.-   16. Lomas-Neira J L, Chung C-S, Wesche D E, Perl M, Ayala A. In vivo    gene silencing (with siRNA) of pulmonary expression of MIP-2 versus    K C results in divergent effects on hemorrhage-induced,    neutrophil-mediated septic acute lung injury. J Leukoc Biol 2005;    77: 846-53.-   17. Davis M E, Zuckerman J E, Choi C H J, et al. Evidence of RNAi in    humans from systemically administered siRNA via targeted    nanoparticles. Nature 2010; 464: 1067-70.-   18. Griesenbach U, Vicente C C, Roberts M J, et al. Secreted Gaussia    luciferase as a sensitive reporter gene for in vivo and ex vivo    studies of airway gene transfer. Biomaterials 2011; 32: 2614-24.    Example Implementation of Electrospray-Delivery of Nucleic Acids to    Ex Vivo Porcine Airways Using Electrospray

Nucleic acid-based therapies have the potential to provide clinicallymeaningful benefit across a wide spectrum of lung disease. However, invivo delivery remains a challenge. Here we examined the feasibility ofusing electrospray to deliver nucleic acids to both porcine trachealtissue sections and whole lung ex vivo.

The effect of electrospray solution, emitter gauge, flow rate andvoltage on plasmid DNA integrity was examined by analysingsupercoiled:open circle structure ratio by gel electrophoresis. Optimalparameters were used to deliver luciferase DNA and mRNA and siRNA-FITCto tracheal tissue sections. Luciferase mRNA was delivered to wholeporcine lungs ex vivo using a catheter and bronchoscope system.Luciferase activity and fluorescence were analysed by luminometry andmicroscopy respectively.

The incidence of DNA plasmid nicking was greatest in a low salt solutionwithout ethanol compared with 1% and 20% ethanol with salt. From a rangeof emitters tested, a 32 gauge emitter produced the bestsupercoiled:open circle structure ratio, likely because less voltage wasrequired to produce a stable electrospray with this emitter. Lower flowrates also showed a trend towards reduced DNA nicking. GFP DNAelectrosprayed at 5 kV and 6 kV resulted in lower levels of GFPexpression in A549 lung cells following lipofection compared with 3 kVand 4 kV. Optimised parameters of 20% ethanol solution, 32 gaugeemitter, low flow rates and voltages of 3-5 kV, nucleic acid moleculeswere successful for delivery of luciferase DNA and mRNA as well assiRNA-FITC to porcine tracheal tissue sections and for delivery ofluciferase mRNA to whole porcine lungs via bronchoscope. In summary, wereport ex vivo delivery of nucleic acids to porcine lung tissue viaelectrospray and bronchoscopic electrospray delivery of nucleic acid toan ex vivo porcine lung model.

Nucleic acid-based therapies have the potential to provide clinicallymeaningful benefit across a wide spectrum of lung disease. However,despite decades of investigation of viral and non-viral methods ofnucleic acid delivery to the lungs, no treatments have yet been approvedfor clinical use. Issues with adverse effects and efficiency of deliveryhave hampered progress and the airway mucus gel layer remains asignificant barrier to both viral and non-viral vectors (Duncan G A,Jung J, Hanes J, Suk J S. The Mucus Barrier to Inhaled Gene Therapy. MolTher 2016; 24: 2043-2053). In addition to long-standing gene therapystrategies where a gene is delivered to replace a defective gene orprovide a therapeutic effect, emerging techniques such as gene editinghold promise as new treatment approaches. For these techniques it may benecessary to deliver mRNA, short DNA sequences and/or proteins.Therefore, new delivery strategies are needed to address challenges seenwith traditional gene delivery approaches and to ensure that progresswith new techniques can be translated to the lungs.

Non-viral methods of gene delivery, including chemical and physicalmethods, have benefits as they are less immunogenic than viraltechnologies and have fewer regulatory hurdles for in vivo use. However,the absence of a successful benchmark therapy involving non-viralvectors indicates that further challenges remain for these applications.Aerosolization has long been viewed as a desirable route for genedelivery to the lungs and can be achieved by several methods. The mostcommon method employs a spray nozzle through which fluid passes and isacted upon by mechanical forces that atomize the liquid. For delivery tothe lung, sonication is widely used whereby high frequent vibrationthrough a nebulizer nozzle plate produce small droplets of a liquid.However, because drugs are inspired, this is a high amount of drug lostand poor targeting of delivery. Furthermore, current aerosol deliverymethods such as jet or ultrasonic nebulisers can cause shear stress tothe DNA resulting in a poor efficiency of gene delivery (Gomes dos ReisL, Svolos M, Hartwig B, Windhab N, Young P M, Traini D. Inhaled genedelivery: a formulation and delivery approach. Expert Opin Drug Deliv2017; 14: 319-330.1). The ability to deliver nucleic acid-based drugs ina non-viral, targeted manner to the airways in a way that preservesactivity of the molecules is therefore desirable.

Electrospray, also known as electrostatic spray, is an alternativemethod of atomizing a liquid that is well-established in many fieldsincluding soft ionisation mass spectrometry. An electrospray occurs whentiny quantities of electrical charge are applied to a fluid as it passesthrough a conducting emitter such as a needle (Jaworek A, Sobczyk A T.Electrospraying route to nanotechnology: An overview. J Electrostat2008; 66: 197-219). The potential difference generated between thecharged solution and a ground electrode generates an electric fielddrawing the liquid towards the ground electrode. The meniscus generatedforms a characteristic cone, known as a ‘Taylor’ cone. When the voltagethreshold overcomes the surface tension of the solution, the tip of theTaylor cone dissociates, or atomizes, into droplets forming either ajet, or a plume of charged microdroplets. While other methods of spraygeneration utilise gravity and droplets decelerate once formed, within acertain distance, the electrospray droplets accelerate towards a surface(Rosell-Llompart J, Fernández de la Mora J. Generation of monodispersedroplets 0.3 to 4 μm in diameter from electrified cone jets of highlyconducting and viscous liquids. J Aerosol Sci 1994; 25: 1093-1119). Inaddition, because the droplets are charged, an electrospray is morecontrollable than other forms of spray and therefore lends itself totargeted delivery. These features have led some investigators to examineelectrospray as a method for gene delivery although reports are few andinconsistent, possibly due to significant differences betweenelectrospray configurations, solutions and parameters. While successfuldelivery of plasmid DNA has been reported in cultured A549 lung cellsand mouse skin using electrospray, Zeles-Hahn et al. did not observetransfection of luciferase plasmid DNA in a study of tracheal/bronchialepithelial cells cultured at air-liquid interface (Boehringer S, RuzgysP, Tamo L, Satkauskas S, Geiser T, Gazdhar A, Hradetzky D. A newelectrospray method for targeted gene delivery. Scientific Reports 2018;8:4031; and Zeles-Hahn M G, Lentz Y K, Anchordoquy T J, Lengsfeld C S.Effect of electrostatic spray on human pulmonary epithelial cells. JElectrostat 2011; 69: 67-77). Another electrospray-based technique is‘bio-electrospray’ whereby cells are electrosprayed onto a surface suchas an extracellular matrix-like scaffold in a targeted and controlledmanner. First reported in 2006, bio-electrospray is being investigatedfor applications such as tissue engineering and regenerative medicineand we have previously reported successful bio-electrospray of humanmesenchymal stem cells (McCrea, Z., Arnanthigo, Y., Cryan, S A., O'Dea,S. A novel methodology for bio-electrospraying mesenchymal stem cellsthat maintains differentiation, immunomodulatory and pro-reparativefunctions. Journal of Medical and Biological Engineering. 2017.https://doi.org/10.1007/s40846-017-0331-4).

In order to form a stable Taylor cone and plume during electrospraying,solutions can have optimal conductivity, surface tension and viscositylevels (Boehringer S, Ruzgys P, Tamo L, Satkauskas S, Geiser T, GazdharA, Hradetzky D. A new electrospray method for targeted gene delivery.Scientific Reports 2018; 8:4031). Therefore, solutions typically containlow concentrations of salts as well as solvents such as ethanol.However, the effect of these components on DNA integrity duringelectrospraying has not been examined. Here we report studies ofelectrospray parameters for DNA solutions and successful delivery ofplasmid DNA, mRNA and siRNA to porcine tracheal explant tissue culturedat an air-liquid interface using low voltage electrosprays with abench-top instrument. We subsequently used a catheter-based electrospraydevice to assess the feasibility of electrospray atomisation for thedelivery of nucleic acids directly to the lung in a targeted manner, viabronchoscopy, in a porcine ex vivo lung model.

Materials and Methods

Cell Culture

A549 human lung cell line (Cat. No. 86012804, Sigma-Aldrich, Wicklow,Ireland) was routinely cultured in DMEM (Gibco, Thermo FisherScientific, Dublin, Ireland) supplemented with 5% fetal bovine serum and2 mM L-glutamine (Gibco).

Electrospray Studies

The electrospray device comprised a silent air compressor, syringe pump,laser and camera for visualization of the Taylor cone and plume and acontrol unit (Avectas, Dublin, Ireland). For DNA integrity studies,pEGFP plasmid (Clontech Laboratories, Saint-Germain-en-Laye France)solutions were sprayed into a 24-well cell culture plate (Costar,Sigma-Aldrich) that contained the ground electrode. DNA was diluted in alow salt solution (137 nM NaCl, 1.47 nM KH₂PO₄, 8.10 nM Na₂HPO₄, 2.68 nMKCl), 1% ethanol/H₂O or 20% ethanol/H₂O, all chemicals fromSigma-Aldrich. DNA quantification was carried out using the Nanodrop(Thermo Fisher Scientific). DNA samples were run on a 1% electrophoresisgel, stained with ethidium bromide and scanned using a Gel Doc™ XRSSystem and Quantity One® software (Bio-Rad Laboratories, Watford, UK).

Lipofection

Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) was used totransfect plasmid DNA into A549 cells according to manufacturer'sinstructions. GFP expression was determined using an Accuri flowcytometer (BD Biosciences, Wokingham, UK) 24 hr post-transfection.

Electrospray Delivery to Cultured Porcine Tracheal Explants

Pig lungs were obtained from a local abattoir. Cold ischaemic time waslimited to 90 min. The trachea was dissected into sections approximately20×50 mm, rinsed with phosphate-buffered saline (PBS) and 3-4 washcycles in 1:1 RPMI 1640:DMEM, 200 units/ml penicillin, 200 μg/mlstreptomycin, 2.5 μg/ml amphotericin, 50 μg/ml gentamicin (allSigma-Aldrich) were performed. Each wash cycle included changing washmedia, 10 minutes of agitation on a cell shaker and 1 hr incubation at37° C., 5% CO₂. Tracheas were cut into 10 mm² segments, includingepithelium, mucosa and cartilage. Tissue segments, epithelial layerfacing upwards, were placed onto 5 mm high sterile agarose (1% w/v)plugs in 12-well plates (Costar, Sigma-Aldrich). Culture media (washmedia plus 10 mM L-glutamine and 10% FBS) was added so that the basalsection of the tissue section was submerged. Explants were incubatedovernight at 37° C., 5% CO₂, in a humidified atmosphere. For delivery,the electrospray emitter was positioned above a grounded base plate, 15mm from the epithelial surface of the tissue. Voltage was adjustedbetween 3-5 kV to achieve a stable Taylor cone and diffuse plumedepending on environmental conditions (humidity/temperature). Nucleicacids were resuspended in delivery solution 20% ethanol/H₂O. Porcinetissues were placed epithelial surface up, in the centre of a baseplate, below the electrospray emitter and nucleic acid solutions weredelivered over 2-3 minutes. For pGLuc (New England Biolabs, Ipswich,Mass., USA), three doses 5 μg were delivered over three consecutivedays. For luciferease mRNA (TriLink Biotechnologies, San Diego, Calif.,USA) and siRNA-FITC (Thermo Fischer Scientific) the total treatment wasadministered with a single spray. Following nucleic acid delivery, thetissue segments were placed on agarose plugs in fresh culture medium.For pGLuc and luciferase mRNA activity, supernatant was analysed usingthe Biolux™ Gaussia Luciferase Assay (New England Biolabs) according tomanufacturer's instructions. Fluorescence was analysed using the OlympusCKX41 microscope (Olympus, Stansfield, UK).

Electrospray Delivery to Whole Porcine Lung Ex Vivo

The porcine heart-lung block was prepared by cannulating the superiorvena cava and perfusing culture medium supplemented with 7% albumin and0.5% dextran through the pulmonary vasculature with a peristaltic pump(Masterflex, Gelsenkirchen, Germany). The trachea was intubated andventilated (IPAP 14; EPAP 4; FiO2 21%) using a NIPPY 2 ventilator(RespiCare Ltd., Swords, Ireland). The lungs were cleared of debris byinstilling 10 ml aliquots of 0.9% saline and suctioning. Theelectrospray catheter system was composed of an electrospray catheter,power pack and a syringe pump (Avectas). Prior to bronchoscopyelectrospray delivery, a target endobronchial area was selected andmarked with 3 dots to form a target using SPOT endoscopic marker (GISupply, Camp Hill, Pa., USA).

Statistics

Two-tailed, unpaired T-Tests were used for statistical analysis usingGraphpad Prism version 7.03 for Windows (GraphPad Software, La Jolla,Calif., USA).

Results

Effect of Electrospray Solution Composition on Plasmid DNA Integrity

Zeles-Hahn et al. appear to have used various solutions containing 20ng/μl GFP DNA without a solvent, the equivalent of a 25 gauge emitter, aflow rate of 200 μl/min and applied voltages of 3-7 kV in negative modeat a distance of 25 mm from ground (Zeles-Hahn M G, Lentz Y K,Anchordoquy T J, Lengsfeld C S. Effect of electrostatic spray on humanpulmonary epithelial cells. J Electrostat 2011; 69: 67-77). Theyreported dripping, stable cone-jet and whipping cone electrosprayprofiles at −3 kV, −6 kV and −7 kV respectively. Boehringer et al. useda sucrose solution containing 100 ng/μl GFP DNA without a solvent, a 29gauge emitter, flow rate of 20 μl/min, applied voltages of 3 kV inpositive mode at distances of 3-6 mm (McCrea, Z., Arnanthigo, Y., Cryan,SA., O'Dea, S. A novel methodology for bio-electrospraying mesenchymalstem cells that maintains differentiation, immunomodulatory andpro-reparative functions. Journal of Medical and Biological Engineering.2017. https://doi.org/10.1007/s40846-017-0331-4). For the present study,we aimed to further study electrospray parameters for transfection ofairway tissue and to analyse the effect of these parameters onelectrospray mode and DNA integrity. For these studies, we used positivemode electrospray as we found this more conducive than negative mode forthe formation of stable sprays (data not shown). Controlnon-electrosprayed samples were taken either before loading into theelectrospray system or generated by allowing solutions to drip throughan emitter without the application of an electrical charge andcollecting in a tissue culture plate from which they were retrieved foranalysis (FIG. 21 a ). When an electric charge was applied to theemitter, the DNA solution formed a classic Taylor cone and plume andthese are referred to as ‘Spray’ samples (FIG. 21 b ).

Covalently closed circular DNA will adopt a supercoiled topology. Asingle break on one strand of the molecule will result in the release ofthe supercoiled form into an open circle form. Another cleavage on theopposite strand will result in a linear DNA form. Further DNA nicks willresult in fragmentation of the DNA. Any of these events could lead toreduced transfection efficiency.

Three solutions containing low concentration salts or ethanol wereselected and their effect on DNA integrity during electrospray wasdetermined. DNA (pEGFP, 100 ng/μl) was resuspended in low salt solution,1% ethanol/H₂O or 20% ethanol/H₂O. Solutions were delivered at flowrates of 5 μl/min for 5 min into empty tissue culture plates andretrieved for analysis. All solutions achieved plume electrospray mode,however voltages of approximately 4 kV were typically required for thelow salt and 1% ethanol solutions compared with 3 kV for the 20% ethanolsolution. We also observed that the low salt and 1% ethanol solutionshad narrower plumes and were less stable over time compared with the 20%ethanol solution. There was an increase in open circle form of theplasmid in the sprayed low salt solution compared with the ethanolsolutions indicating increased single strand nicking (SSN) in the lowsalt solution (FIG. 21 c ).

Effects of Emitter Gauge and Flow Rate

Emitter gauge and solution flow rates are two further parameters thataffect Taylor cone formation and the ability to generate a stableelectrospray. We therefore used the low salt solution to examine whetherthese two parameters were contributing to the nicking observed in theDNA plasmid.

Plasmid DNA in low salt solution was electrosprayed at four differentflow rates (40 μl/min, 20 μl/min, 10 μl/min and 5 μl/min) through sixdifferent sized emitters (22, 23, 25, 27, 30, 32 gauge). For eachemitter gauge tested, each flow rate was compared to the corresponding40 ul/min drip control. While there was no statistically significantdifference in the supercoiled: open circular ratio between the samples,there was a trend towards the 32 ga emitter preserving the highestlevels of supercoiled plasmid at all flow rates (FIG. 22 a ).

The voltage required to form a stable cone-jet electrospray for eachemitter was noted and it was observed that higher voltages were requiredfor the wide internal diameter emitters (22 ga) compared to the narrowerinternal diameter emitters (32 ga) (FIG. 22 b ). These results suggestthat although the larger internal diameter emitters caused more plasmidnicking, this was likely due to the higher voltages required to achievea Taylor cone and plume electrospray.

Effect of High Voltage

We next examined the effects of voltage on DNA nicking. For theseexperiments the resistance was kept constant and the voltage wasincreased in order to assess the effect of current on DNA nicking. Thevoltages examined were 3 kV, 4 kV, 5 kV and 6 kV. Flow rates of 60 and15 μl/min and emitter gauges of 22 ga and 32 ga were tested. Plasmid DNAwas electrosprayed in low salt solution.

Firstly, the effect of these parameters on spray mode was observed byeye (FIG. 23 ). At 3 kV the potential was not high enough to generate aspray at either flow rate or with either emitter gauge. At 4 kV, forboth flow rates, a spray was generated with the 32 ga emitter but notthe 22 ga emitter, which remained in drip mode. At 5 kV, for both flowrates, an unstable spray that alternated between drip and spray wasobserved with the 22 ga emitter while a stable spray was generated withthe 32 ga emitter. At 6 kV the electrical potential was high enough togenerate a continuous stable electrospray with both emitter sizes andboth flow rates.

DNA nicking was next examined at each voltage, flow rate and emittergauge. Firstly, using a 32 ga emitter, an increase in plasmid nickingwas observed with increasing voltage at both 60 and 15 μl/min flowrates, as indicated by an increase in the level of open-circle plasmidDNA (FIG. 23 a ). When the flow rate was kept constant at 60 μl/min andthe voltage was increased, there was no significant difference insupercoiled: open circular ratio between 22 ga or 32 ga emittersindicating that emitter gauge per se does not affect DNA integrity (FIG.23 b ). However, there was increased plasmid nicking as the voltageincreased. There was a significant increase in plasmid nicking using the22 ga emitter at 6 kV compared to 3 kV (p=0.0003). There was also asignificant increase in plasmid nicking using the 32 ga emitter at 5 kVcompared to 3 kV (p=0.002). These results indicate that high voltage, ormore specifically the higher current that this equates to, is thecausative factor of DNA nicking during electrospray.

To determine the consequence of plasmid nicking on DNA expression, aplasmid encoding for green fluorescent protein (pGFP) was subjected toelectrospray and subsequently transfected into A549 lung epithelialcells by lipofection. Equal concentrations of DNA were lipofected andthe transfection efficiency was determined. Increased levels of DNAnicking correlated with decreased levels of GFP expression (FIG. 23 c ).This indicates that single strand nicking of plasmid DNA induced by highvoltage leads to open-circular confirmation and ultimately a reductionin downstream protein expression.

Electrospray Delivery of Plasmid DNA, mRNA and siRNA to Cultured PorcineTracheal Explants

Having examined the effects of various electrospray parameters onintegrity of nucleic acids, we then went on to assess the feasibility ofdelivery to airway tissue. Two ex vivo model systems were used: culturedporcine tracheal explants and whole porcine lung. Various reporterplasmids have been used in studies aimed at developing vectors for genetherapy for airway diseases. A secreted Gaussia princeps luciferase(GLuc) reporter gene has been shown to be a sensitive reporter inpre-clinical studies of gene transfer in cystic fibrosis models so thisplasmid (pGLuc) was used for studies here (Griesenbach U, Vicente C C,Roberts M J, Meng C, Soussi S, Xenariou S, Tennant P, Baker A, Baker E,Gordon C, Vrettou C, McCormick D, Coles R, Green A M, Lawton A E,Sumner-Jones S G, Cheng S H, Scheule R K, Hyde S C, Gill D R, Collie DD, McLachlan G, Alton E W. Secreted Gaussia luciferase as a sensitivereporter gene for in vivo and ex vivo studies of airway gene transfer.Biomaterials. 2011 32(10):2614-24). Based on results above, thefollowing parameters were deemed best for maintaining integrity ofnucleic acid molecules: 20% ethanol/H₂O as delivery solution, 32 gaemitter and low flow rates.

Porcine tracheal explants were electrosprayed with 5000 ng pGLuc (833ng/μl; flow rate 3 μl/min; duration 2 min) once each day for 3consecutive days and tissues were then cultured for a further 48 hr(FIG. 24 a ). 20 μl of culture media was sampled from each well andluciferase activity was quantified. There was a significant increase inluciferase activity (p=0.0002) in the media of pGLuc electrosprayedtissue compared to the media from electrosprayed controls (FIG. 24 b ).

We next evaluated electrospray delivery of RNA molecules. mRNA encodingfor luciferase was performed on porcine tracheal tissue. Luciferase mRNA(10 μg) was electrosprayed onto the trachea tissue (700 ng/μl; flow rate4.8 μl/min; duration 3 min) and incubated for 48 hours. Deliverysolution without mRNA was electrosprayed as a negative control.Quantification of luciferase activity showed a significant increase inluminescence expression (p=0.023) in the mRNA electrosprayed samplescompared to control explants (FIG. 25 a ).

siRNA-FITC (20 μl 100 μM) was delivered to the trachea explant (100 μM;flow rate 10 μl/min; duration 2 min) and compared to a deliverysolution-only negative control. Following delivery, the epithelial layerwas microdissected and examined under a fluorescent microscope. FITCfluorescence was evident in tissue that were electrosprayed with siRNAbut not in control tissue (FIG. 25 b ). In addition, tracheal segmentswere cryosectioned in the transverse plane, allowing visualisation ofsiRNA within the tissue (FIG. 25 c ).

Bronchoscopic Electrospray Delivery of mRNA to Whole Porcine Lung ExVivo

We next examined whether electrospray could be used to deliver nucleicacids to the airways within whole lungs. An apparatus was set upcomprising catheter, bronchoscope and electrospray equipment as before(FIG. 20 ). Dissected lungs were obtained and prior to bronchoscopicelectrospray delivery, a target endobronchial area was selected andmarked with 3 dots to form a target using SPOT endoscopic marker. Theelectrospray catheter was then inserted into the working port of abronchoscope, positioned 15 mm from the pre-marked target and deliversolution containing mRNA was delivered to the centre of the dots. 10 μl(3 μg) mRNA-GLuc (300 ng/μl; flow rate 100 μl/sec; duration 0.1 sec) wasdelivered to target areas which were then resected, washed and culturedat air liquid interface. Luciferase activity was measured at 48 hrpost-delivery. A significant (p<0.0001) increase in luciferase activitywas detected in luciferase mRNA-treated samples compared to controls(FIG. 26 ).

Discussion

Nucleic acid based therapies have significant disease modifyingpotential. As such, there has been much interest in developingtechniques that successfully deliver these molecules in vivo. Multipleviral and non-viral vectors have been utilised for this aim, but successhas been limited to date. Challenges of cytotoxicity, immunogenicity andtransfection inefficiency have hampered progress. Improved safetyprofiles with non-viral vectors have increased interest in thesetechniques. However, technologies to improve transfection efficacy areneeded if non-viral vector therapies are to have a clinically meaningfuleffect. This study is the first, to our knowledge, to report thesuccessful electrospray delivery of RNA or DNA molecules to lung tissue.Porcine lung was selected as a target for gene delivery in view of itsextensive use in translational research and its close approximation ofthe human lung for bronchoscopic intervention (Rogers C S, Abraham W M,Brogden K a, Engelhardt J F, Fisher J T, McCray P B et al. The porcinelung as a potential model for cystic fibrosis. Am J Physiol Lung CellMol Physiol 2008; 295: L240-L263; and Judge E P, Hughes J M L, Egan J J,Maguire M, Molloy E L, O'Dea S. Anatomy and bronchoscopy of the porcinelung: A model for translational respiratory medicine. Am. J Respir. CellMol. Biol. 2014; 51: 334-343). Furthermore, the epithelial surface ofthe lung provides an accessible target for non-invasive (inhalation) andsemi-invasive (bronchoscopy) drug delivery. Electrospray atomisation hasthe potential to be utilised as a drug delivery platform for bothmodalities.

Electrospray ionisation is a vector-free delivery system that has thepotential to overcome multiple barriers associated with inefficienttransfection. This technique has previously been reported tosuccessfully transfect plasmid DNA in vitro and to mouse skin(Boehringer S, Ruzgys P, Tamo L, Satkauskas S, Geiser T, Gazdhar A,Hradetzky D. A new electrospray method for targeted gene delivery.Scientific Reports 2018; 8:4031; Ikemoto K, Sakata I, Sakai T. Collisionof millimeter droplets induces DNA and protein transfection into cells.Sci Rep 2012; 2: 1-5; and Lee Y H, Wu B, Zhuang W Q, Chen D R, Tang Y J.Nanoparticles facilitate gene delivery to microorganisms via anelectrospray process. J Microbiol Methods 2011; 84: 228-233). Here wereport the successful electrospray ex vivo delivery of plasmid DNA, mRNAand siRNA molecules. In this study, protein expression followingtransfection was identified, indicating that biological integrity ofnucleic acid solutions is maintained during the electrospray process.

Zeles-Hahn et al. failed to achieve transfection of luciferase DNA inthe form of naked plasmid DNA, lipid/DNA complexes,polyethyleneimine/DNA complexes and poly L-lysine/DNA complexes intohuman airway epithelial cells (Zeles-Hahn M G, Lentz Y K, Anchordoquy TJ, Lengsfeld C S. Effect of electrostatic spray on human pulmonaryepithelial cells. J Electrostat 2011; 69: 67-77). They do not appear tohave used a solvent and this may have necessitated high voltages inorder to cause the solution to break up into a spray. Such voltages maydamage the structure of the DNA plasmid and they report an increase inopen circle and fragmented DNA at 6 kV and 7 kV.

The process of electrospray atomisation involves the generation of anelectric field between a charged solution and a grounded collector. Asthe electromagnetic field overcomes surface tension, the solution isdispersed into nano-sized particles that move towards a collector athigh velocity. The characteristics of electrospray generatedparticles—size, charge, velocity and direction—may benefit delivery bypotentially addressing factors that reduce the efficiency of nucleicacid delivery to tissue. Electrospray may create conditions whereparticles pass through a cell membrane by kinetic force, as happens witha ‘gene gun’ (Zhang D, Das D B, Rielly C D. Potential ofmicroneedle-assisted micro-particle delivery by gene guns: a review.Drug Deliv 2013; 7544: 571-587). This may also be achieved by theinteraction of charged particles with membrane-bound voltage gates orion channels. In addition, the electric field associated with anelectrospray may induce the transient formation of pores within the cellmembrane, known as electroporation (Lambricht L, Lopes A, Kos S, SersaG, Préat V, Vandermeulen G. Clinical potential of electroporation forgene therapy and DNA vaccine delivery. Expert Opin Drug Deliv 2015;5247: 295-310).

Delivery of plasmid DNA to tissue can be challenging because the largesize of these molecules can negatively impact upon cellular uptake. Inaddition, cellular uptake of DNA does not guarantee protein expressionas nucleolar relocation must occur before protein encoding can commence.We hypothesised that the delivery of smaller molecules, such as mRNA,would increase the efficacy of protein expression by removing barriersassociated with molecular size and ribosomal transcription. mRNA mayalso have therapeutic advantages over DNA, where factors such astransient protein expression may be an important consideration in drugdevelopment. In our study, we did not directly compare transfectionefficiency of mRNA to plasmid DNA, however a 2 log increase inluciferase activity was seen with both plasmid DNA and mRNA compared tocontrols.

Gene regulation, and therefore disease modulation, by siRNA moleculesalso have great potential for treating conditions such as cancer (GolanT, Khvalevsky E Z, Hubert A, Gabai R M, Hen N, Segal A et al. RNAitherapy targeting KRAS in combination with chemotherapy for locallyadvanced pancreatic cancer patients. Oncotarget 2015; 6: 24560-24570).We have shown that fluorescent labelled siRNA molecules can be deliveredto the epithelial and mucosal layers of lung tissue by electrospray.While siRNA function was not assessed in the study, this therapeuticapproach would have the potential to downregulate disease pathwaysduring infection, inflammation or oncogenesis (Zamora M R, Budev M,Rolfe M, Gottlieb J, Humar A, DeVincenzo J et al. RNA interferencetherapy in lung transplant patients infected with respiratory syncytialvirus. Am J Respir Crit Care Med 2011; 183: 531-538; Lomas-Neira J L,Chung C-S, Wesche D E, Perl M, Ayala A. In vivo gene silencing (withsiRNA) of pulmonary expression of MIP-2 versus K C results in divergenteffects on hemorrhage-induced, neutrophil-mediated septic acute lunginjury. J Leukoc Biol 2005; 77: 846-53; and Davis M E, Zuckerman J E,Choi C H J, Seligson D, Tolcher A, Alabi C A et al. Evidence of RNAi inhumans from systemically administered siRNA via targeted nanoparticles.Nature 2010; 464: 1067-1070).

REFERENCES

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Experiment 3—Porous Tip Emitter Head

To investigate the viability of using a porous tip, a felt tip part of awriting instrument (e.g., Copic markers) was tested. A high-voltageelectrical connection was made with a copper washer. Testing equipmentincluded the electrospray device base rig with a power supply, a flatplate counter electrode mounted on a lab jack. For performancediagnosis, a microscopic a VIMS videoscope was placed near the tip toprovide illumination.

For an experiment, 77% ethanol was supplied from the proximal end of thewriting instrument as illustrated in FIG. 28 . FIGS. 29 and 30 show theelectrospray formation using the porous tip. Experiment results showthat the porous tip can establish a uniform and stable spray at supplyvoltages ranging from 2.0 to 2.9 kV at tip-to-plate distances of 10 to15 mm. Above 2.9 kV, a multijet mode where two or more distinct jets areobserved was established. The uniform spray was demonstrated by a cleardeposition of the liquid on the counter electrode. Placing a plasticinsert onto the flat plate collector showed similar behavior observedwith the steel needle, i.e., the spray was deflected by the insert anddeposited around it. A ring deposition pattern was observed indicatingthat the electric field lines were deflected by the plastic insert.

In another variation of the delivery platform with the porous tip, aclass 2 laser light source may be used to illuminate the plume as themonochromatic and collimated light source can provide betterillumination. In another experiment, it was demonstrated that theelectrospray can supply a stable cone-jet mode spray for about 2 minutesand 52 seconds when filled with 100 μL of liquid and subject to a supplyvoltage of 2.7 kV with a tip-to-plate distance of 6 mm or about 5minutes and 32 seconds with a tip-to-plate distance of 26 mm. At atip-to-plate distance of 26 mm, stable cone-jet mode was observed from2.8 kV to 3.4 kV while above 3.7 kV, multijet mode started to appear.Observation of the electrode plate showed that the coverage pattern hada larger diameter at a tip-to-place distance of 26 mm than at 6 mm. Nocoverage was observed with no spray. FIGS. 31-33 show experimentalresults for various supply voltages using the porous tip with 77%ethanol at different tip-to-plate distances.

Various porous tips with different configurations were also tested.Specifically, 4 different nibs from Copic writing instrument products:standard super brush, standard fine, brush, and multiliner were tested.The brush and multiliner nibs had metal connections, but the standardfine and super brush needed the connectors to be made. Among the onesthat were tested, the standard fine demonstrated a stable con-jetformation. The testing procedure was saturating the nib and addingsolution to induce a flow, and then setting voltage at about 2.5 kV andtaking a picture to see the response.

In this experiment, four different solutions were tested: a) 100%ethanol, b) deionized water, c) 0.25% methylene blue+0.01% ammoniumacetate, and d) 0.25% methylene blue+1% ethanol. The test was made at aconstant tip-to-plate distance of 11 mm. Summarizing the results,ethanol sprayed well as expected. The deionized water showed a jetligament at 4.4 kV without a plume. However, when the counter electrodewas removed and the solution sprayed into free space, an evidence ofplume formation was observed for deionized water. The ethanol anddeionized water tests are illustrated in FIGS. 34 and 35 , respectively.The 0.25% methylene blue and 0.01% ammonium acetate solutiondemonstrated spray formation over a voltage range of 2.8 kV-3.1 kV. Theobserved spray pattern was a plume with a ligament at the center. Below2.8 kV, there was not enough energy to draw the solution and no spraywas observed. Above 3.1 kV, the spray appeared to pulsate or becomesporadic with no sign of a stable plume. Within the spray voltage range,the spray can be controlled on and off by the foot switch as illustratedin FIG. 36 . When the foot switch was latched off, the spray instantlystopped. When latched on, there was a delay as the system ramped up tothe set voltage. Further, the spray range was sensitively affected bythe position of the counter electrode, e.g., moving the position of theplate can require a small adjustment of the voltage to achieve a stablespray. Moreover, unlike deionized water, no spray was observed with nocounter electrode for methylene blue. The 0.25% methylene blue and 1%ethanol solution was more difficult to spray. It showed signs of aplume, but unstable and pulsating at times as illustrated in FIG. 37 .In addition, the 0.25% methylene blue and 0.01% ammonium acetatesolution was tested with a super fine brush nib, but the configurationdid not demonstrate a stable spray.

In another experiment, the catheter coupled to a porous tip emitter headwas tested with methylene blue and demonstrated a stable electrospray at7.5 kV with the counter electrode present. The methylene blue deliverytest was conducted within a bronchoscope onto the bench, and the spraydelivery was achieved in dry and wet conditions for tip-to-platedistances from 9 to 16 mm. FIG. 38 illustrates an exemplary benchtoptest setup of the catheter coupled to a porous tip emitter head. Inaddition, the methylene blue delivery test was conducted to ex vivoventilated porcine lungs. FIGS. 39 and 40 show the ex vivo testing ofthe catheter with a porous tip to ventilated porcine lungs. To achievestable spraying, the metal shroud was covered with parafilm asillustrated in FIG. 40 . A tip-to-scope distance of 12 mm was used forthis experiment. From this experiment, 100% (4 out of 4) effectivenessin trachea was observed.

In yet another experiment, the methylene blue delivery test wasconducted to ex vivo ventilated porcine lungs with the tip-to-scopedistance varying from 3 to 13 mm. 79% (29 out of 37) effectiveness intrachea was achieved, and comparable effectiveness was achieved in alltip-to-scope distances. It was observed that the spray consistentlypropelled in the forward direction with no wall attraction. A goodelectrical isolation was observed as well, and no current exceeding 100μA was produced.

In the descriptions above and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

The subject matter described herein can be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The implementations set forth in the foregoingdescription do not represent all implementations consistent with thesubject matter described herein. Instead, they are merely some examplesconsistent with aspects related to the described subject matter.Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations can be provided in addition to those set forth herein.For example, the implementations described above can be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flows depicted in theaccompanying figures and/or described herein do not necessarily requirethe particular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of the followingclaims.

What is claimed is:
 1. An apparatus comprising: a catheter defining afluidic channel and having a distal opening, wherein the fluidic channelis configured to receive a metered volume of fluid, and wherein thefluid contains ethanol at greater than 5% concentration; an electrodewithin the fluidic channel and spaced a distance from the distal openingof the catheter; a porous tip disposed within the fluidic channel anddisposed near the distal opening of the catheter, the porous tipcomprising a material that is absorbent and fibrous, and wherein theporous tip is arranged in a cone shape geometry; a protrusion disposednear the distal opening of the catheter and surrounding an outer surfaceof the catheter; a thermoplastic wrap that encloses the protrusion toprovide sealing and electrical isolation, such that the apparatusproduces no current exceeding 100 μA; and a pump coupled to thecatheter, wherein the pump is configured to provide the metered volumeof fluid to the fluidic channel, wherein the metered volume of fluid isbetween 1 and 10 microliters of fluid per actuation of the pump, whereinthe catheter is arranged to prevent direct contact between any electrodeof the apparatus and tissue.
 2. The apparatus of claim 1, furtherincluding a conductive sheath on an exterior of the catheter configuredto couple to a ground, the catheter separating the fluidic channel andthe conductive sheath.
 3. The apparatus of claim 2, further including abiocompatible cover enclosing the conductive sheath between thebiocompatible cover and the catheter.
 4. The apparatus of claim 3,wherein the biocompatible cover extends less than an entire length ofthe catheter, and a distal end of the catheter is exposed.
 5. Theapparatus of claim 4, wherein the catheter has an inner diameter ofabout 0.5 mm and an outer diameter of about 1.4 mm; the distance betweenthe electrode and the distal opening is about 100 mm; the biocompatiblecover has an exterior diameter of about 2.05 mm, and the catheterextends about 50 mm beyond the biocompatible cover at the distal end. 6.The apparatus of claim 1, further including a wire extending through aportion of a length of the catheter and within the fluidic channel, thewire including an insulation layer and an exposed distal portion, theexposed distal portion forming the electrode.
 7. The apparatus of claim1, wherein the electrode, when carrying a charge, imparts the charge tofluid traveling through the fluidic channel, the charge imparted withinthe fluidic channel so that, when charged fluid reaches a fluid/airinterface near the distal opening of the catheter, the charged fluidforms charged droplets that disperse.
 8. The apparatus of claim 1,wherein the porous tip includes a felt material.